HOW ENZYMES

WORK

ENZYMES SPEED UP CHEMICAL REACTIONSENZYMES SPEED UP CHEMICAL REACTIONS

Enzymes are biological catalysts ndash substances that speed a reaction without being altered in the reaction

Most enzymes are proteins

Enzymes are essential for life

Model of the surface of an enzyme

Enzymes

1048698 Cofactors

1048698 Coenzymes

1048698 Holoenzyme

1048698 Apoenzyme

bull Body conditions(temperature pressure etc) not good for reaction

bull Only enzymes can catalyse the reactions in this conditions

bull A special environment inside enzymes for reaction ACTIVE SITE

bull Molecule binds active site SUBSTRATE

How Enzymes Work

Enzymes Lower a Reactionrsquos Activation Energy

Each reaction has a transition state where thesubstrate is in an unstable short-livedchemicalstructural state

Free Energy of Activationis symbolized by ΔGDagger

Enzymes actby lowering the freeenergy of the transitionstate

Enzymes speed up metabolicreactions by lowering energy barriers

Enzyme speed reactions by lowering EAndash The transition state can be reached at moderate temperatures

Enzymes do not change delta Gndash It speed-up reactions that would occur eventually

Because enzymes are so selective they determine which chemical processes will occur at any time

Enzymes lower the free energy of activation by binding the transition state of the reaction better than the substrate

The enzyme must bind the substrate in the correct orientation otherwise there would be no reaction

Not a lock amp key but induced fit ndash the enzyme andor the substrate distort towards the transition state

Induced FitInduced Fit

A change in the shapeshape of an enzymersquos active site

Induced Induced by the substrate

Lock and Key Model

An enzyme binds a substrate in a region called the active site

Only certain substrates can fit the active site

Amino acid R groups in the active site help substrate bind

Enzyme-substrate complex forms

Substrate reacts to form product

Product is released

Enzyme Kinetics

- Kinetics The study of the rate of change

- Enzyme Kinetics Rate of chemical

reactions mediated by enzymes Enzymes can increase reaction rate by favoring or

enabling a different reaction pathway with

a lower activation energy making it easier

for the reaction to occur

Michaelis-Menten kinetics

V0 varies with [S]Vmax approachedasymptoticallyV0 is moles of

productformed per sec when [P]is low (close to zero time)E + SESE + P

Michaelis-Menten Model

V0 = Vmax x[S]([S] + Km)

Michaelis-Menten Equation

Determining initial velocity (when [P] is low)

Steady-state amp pre-steady-state conditions

At equilibrium no net change of [S] amp [P]or of [ES] amp [E]

At pre-steady-state[P] is low (close to zero time) hence V0 for initial

reaction velocity

At pre-steady state we can ignore the back reactions

Michaelis-Menten kinetics (summary)

Enzyme kinetics (Michaelis-Menten Graph) At fixed concentration of enzyme V0 is almost linearly proportional to

[S] when [S] is small but is nearly independent of [S] when [S] is large

Proposed Model E + S ES E + P

ES complex is a necessary intermediateObjective find an expression that relates rate of catalysis to theconcentrations of S amp E and the rates of individual steps

Start with V0 = k2[ES] and derive V0 = Vmax x[S]([S] + Km) This equation accounts for graph dataAt low [S] ([S] lt Km) V0 = (VmaxKm)[S]At high [S] ([S] gt Km) V0 = Vmax

When [S] = Km V0 = Vmax2 Thus Km = substrate concentration at which the reaction rate (V0) is half max

Range of Km values

Km provides approximation of [S] in vivo for many enzymes

Lineweaver-Burk plot (double-reciprocal)

Eadie-Hofstee plot

Hanes-Woolf Plot

Allosteric enzymes

Allosteric enzymes tend to be

multi-sub unit proteins

The reversible binding of an

allosteric modulator (here a

positive modulator M) affects

the substrate binding site

T

T

R

T

[S]

vo

Mechanism and Example of Allosteric Effect

S S

R

R

SS

RS

A

I

T[S]

vo

[S]

vo

(+)

(-) X X

X

R = Relax(active)

T = Tense(inactive)

Allosteric siteHomotropic(+)Concerted

Heterotropic(+)Sequential

Heterotropic(-)Concerted

Allosteric site

Kinetics Cooperation Models

(-)

(+)

(+)

Enzyme Inhibitors

bull Specific enzyme inhibitors regulate enzyme activity and help us

understand mechanism of enzyme action (Denaturing agents are not

inhibitors)

bull Irreversible inhibitors form covalent or very tight permanent bonds with

aa at the active site of the enzyme and render it inactive 3 classes

groupspecific reagents substrate analogs suicide inhibitors

bull Reversible inhibitors form an EI complex that can be dissociated back

to enzyme and free inhibitor 3 groups based on their mechanism of

action competitive non-competitive and uncompetitive

Enzyme Inhibition

Competitive inhibitors

bull Compete with substrate for binding to enzyme

bull E + S = ES or E + I = EI Both S and I cannot bind enzyme at the same time

bull In presence of I the equilibrium of E + S = ES is shifted to the left causing dissociation of ES

bull This can be reversed corrected by increasing [S]

bull Vmax is not changed KM is increased by (1 + IKi)

bull Eg AZT antibacterial sulfonamides the anticancer agent methotrexate etc

Competitive Inhibition

Kinetics of competitive inhibitor

Increase [S] toovercomeinhibition

Vmax attainable

Km is increased

Ki =dissociationconstant forinhibitor

V max unaltered Km increased

Non-competitive Inhibitors

bull Inhibitor binding site is distinct from substrate binding site Can bind to free enzyme E and to ES

bull E + I = EI ES + I = ESI or EI + S = ESI

bull Both EI and ESI are enzymatically inactive

bull The effective functional [E] (and [S]) is reduced

bull Reaction of unaffected ES proceeds normally

bull Inhibition cannot be reversed by increasing [S]

bull KM is not changed Vmax is decreased by (1 + IKi)

Mixed (Noncompetitive) Inhibition

Kinetics of non-competitive inhibitor

Increasing [S] cannotovercome inhibition

Less E availableV max is lowerKm remains the samefor available E

Km unaltered V max decreased

Uncompetitive Inhibitors

bull The inhibitor cannot bind to the enzyme directly but can only bind to the enzyme-substrate complex

bull ES + I = ESI

bull Both Vmax and KM are decreased by (1+IKi)

Uncompetitive Inhibition

Substrate Inhibition

Caused by high substrate concentrations

E + S ES E + PKm

rsquo k2

KS1

+

S

ES21

2

2

][][

][

][

]][[

][

]][[

Sm

m

mSi

KS

SK

SVv

ES

ESK

ES

ESSK

Substrate Inhibition

At low substrate concentrations [S]2Ks1ltlt1 and inhibition is not observed

Plot of 1v vs 1[S] gives a line Slope = Krsquo

mVm

Intercept = 1Vm

][

111

][1

SV

K

Vv

SK

Vv

m

m

m

m

m

Substrate Inhibition

At high substrate concentrations Krsquom[S]ltlt1 and

inhibition is dominant

Plot of 1v vs [S] gives a straight line Slope = 1KS1 middot Vm

Intercept = 1Vm

mSm

S

m

VK

S

Vv

KS

Vv

1

1

][11

][1

1

max][

0][

SmKKS

Sddv

1V Igt0

I=0

1Vm

-1Km -1Kmapp 1[S]

1VIgt0

I=0

1Vm

-1Km-1Kmapp 1[S]

1Vmapp

1VIgt0

I=0

1Vm

-1Km 1[S]

1Vmapp

1V

1Vm

-1Km 1[S]

Competitive Uncompetitive

Non-Competitive Substrate Inhibition

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] onlyand competes with [S]increasing [S] overcomesInhibition by [II]

[II] binds to free [E] or [ES] complex Increasing [S] cannot overcome [II] inhibition

[II] binds to [ES] complex only increasing [S] favorsthe inhibition by [II]

E + S rarr ES rarr E + P + IIdarrEII

larr

uarr

E + S rarr ES rarr E + P + + II IIdarr darrEII + S rarrEIIS

larr

uarr uarr

E + S rarr ES rarr E + P + II darr EIIS

larr

uarr

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Kmrsquo [S] mM

vo

[S] mM

vo

II II

Km [S] mM

Vmax

II

Kmrsquo

VmaxrsquoVmaxrsquo

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax amp Km decreased

II

1[S]1Km

1vo

1 Vmax

II

Two parallellines

II

Intersect at X axis

1vo

1 Vmax

1[S]1Km 1[S]1Km

1 Vmax

1vo

Intersect at Y axis

= Kmrsquo

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

ENZYMES SPEED UP CHEMICAL REACTIONSENZYMES SPEED UP CHEMICAL REACTIONS

Enzymes are biological catalysts ndash substances that speed a reaction without being altered in the reaction

Most enzymes are proteins

Enzymes are essential for life

Model of the surface of an enzyme

Enzymes

1048698 Cofactors

1048698 Coenzymes

1048698 Holoenzyme

1048698 Apoenzyme

bull Body conditions(temperature pressure etc) not good for reaction

bull Only enzymes can catalyse the reactions in this conditions

bull A special environment inside enzymes for reaction ACTIVE SITE

bull Molecule binds active site SUBSTRATE

How Enzymes Work

Enzymes Lower a Reactionrsquos Activation Energy

Each reaction has a transition state where thesubstrate is in an unstable short-livedchemicalstructural state

Free Energy of Activationis symbolized by ΔGDagger

Enzymes actby lowering the freeenergy of the transitionstate

Enzymes speed up metabolicreactions by lowering energy barriers

Enzyme speed reactions by lowering EAndash The transition state can be reached at moderate temperatures

Enzymes do not change delta Gndash It speed-up reactions that would occur eventually

Because enzymes are so selective they determine which chemical processes will occur at any time

Enzymes lower the free energy of activation by binding the transition state of the reaction better than the substrate

The enzyme must bind the substrate in the correct orientation otherwise there would be no reaction

Not a lock amp key but induced fit ndash the enzyme andor the substrate distort towards the transition state

Induced FitInduced Fit

A change in the shapeshape of an enzymersquos active site

Induced Induced by the substrate

Lock and Key Model

An enzyme binds a substrate in a region called the active site

Only certain substrates can fit the active site

Amino acid R groups in the active site help substrate bind

Enzyme-substrate complex forms

Substrate reacts to form product

Product is released

Enzyme Kinetics

- Kinetics The study of the rate of change

- Enzyme Kinetics Rate of chemical

reactions mediated by enzymes Enzymes can increase reaction rate by favoring or

enabling a different reaction pathway with

a lower activation energy making it easier

for the reaction to occur

Michaelis-Menten kinetics

V0 varies with [S]Vmax approachedasymptoticallyV0 is moles of

productformed per sec when [P]is low (close to zero time)E + SESE + P

Michaelis-Menten Model

V0 = Vmax x[S]([S] + Km)

Michaelis-Menten Equation

Determining initial velocity (when [P] is low)

Steady-state amp pre-steady-state conditions

At equilibrium no net change of [S] amp [P]or of [ES] amp [E]

At pre-steady-state[P] is low (close to zero time) hence V0 for initial

reaction velocity

At pre-steady state we can ignore the back reactions

Michaelis-Menten kinetics (summary)

Enzyme kinetics (Michaelis-Menten Graph) At fixed concentration of enzyme V0 is almost linearly proportional to

[S] when [S] is small but is nearly independent of [S] when [S] is large

Proposed Model E + S ES E + P

ES complex is a necessary intermediateObjective find an expression that relates rate of catalysis to theconcentrations of S amp E and the rates of individual steps

Start with V0 = k2[ES] and derive V0 = Vmax x[S]([S] + Km) This equation accounts for graph dataAt low [S] ([S] lt Km) V0 = (VmaxKm)[S]At high [S] ([S] gt Km) V0 = Vmax

When [S] = Km V0 = Vmax2 Thus Km = substrate concentration at which the reaction rate (V0) is half max

Range of Km values

Km provides approximation of [S] in vivo for many enzymes

Lineweaver-Burk plot (double-reciprocal)

Eadie-Hofstee plot

Hanes-Woolf Plot

Allosteric enzymes

Allosteric enzymes tend to be

multi-sub unit proteins

The reversible binding of an

allosteric modulator (here a

positive modulator M) affects

the substrate binding site

T

T

R

T

[S]

vo

Mechanism and Example of Allosteric Effect

S S

R

R

SS

RS

A

I

T[S]

vo

[S]

vo

(+)

(-) X X

X

R = Relax(active)

T = Tense(inactive)

Allosteric siteHomotropic(+)Concerted

Heterotropic(+)Sequential

Heterotropic(-)Concerted

Allosteric site

Kinetics Cooperation Models

(-)

(+)

(+)

Enzyme Inhibitors

bull Specific enzyme inhibitors regulate enzyme activity and help us

understand mechanism of enzyme action (Denaturing agents are not

inhibitors)

bull Irreversible inhibitors form covalent or very tight permanent bonds with

aa at the active site of the enzyme and render it inactive 3 classes

groupspecific reagents substrate analogs suicide inhibitors

bull Reversible inhibitors form an EI complex that can be dissociated back

to enzyme and free inhibitor 3 groups based on their mechanism of

action competitive non-competitive and uncompetitive

Enzyme Inhibition

Competitive inhibitors

bull Compete with substrate for binding to enzyme

bull E + S = ES or E + I = EI Both S and I cannot bind enzyme at the same time

bull In presence of I the equilibrium of E + S = ES is shifted to the left causing dissociation of ES

bull This can be reversed corrected by increasing [S]

bull Vmax is not changed KM is increased by (1 + IKi)

bull Eg AZT antibacterial sulfonamides the anticancer agent methotrexate etc

Competitive Inhibition

Kinetics of competitive inhibitor

Increase [S] toovercomeinhibition

Vmax attainable

Km is increased

Ki =dissociationconstant forinhibitor

V max unaltered Km increased

Non-competitive Inhibitors

bull Inhibitor binding site is distinct from substrate binding site Can bind to free enzyme E and to ES

bull E + I = EI ES + I = ESI or EI + S = ESI

bull Both EI and ESI are enzymatically inactive

bull The effective functional [E] (and [S]) is reduced

bull Reaction of unaffected ES proceeds normally

bull Inhibition cannot be reversed by increasing [S]

bull KM is not changed Vmax is decreased by (1 + IKi)

Mixed (Noncompetitive) Inhibition

Kinetics of non-competitive inhibitor

Increasing [S] cannotovercome inhibition

Less E availableV max is lowerKm remains the samefor available E

Km unaltered V max decreased

Uncompetitive Inhibitors

bull The inhibitor cannot bind to the enzyme directly but can only bind to the enzyme-substrate complex

bull ES + I = ESI

bull Both Vmax and KM are decreased by (1+IKi)

Uncompetitive Inhibition

Substrate Inhibition

Caused by high substrate concentrations

E + S ES E + PKm

rsquo k2

KS1

+

S

ES21

2

2

][][

][

][

]][[

][

]][[

Sm

m

mSi

KS

SK

SVv

ES

ESK

ES

ESSK

Substrate Inhibition

At low substrate concentrations [S]2Ks1ltlt1 and inhibition is not observed

Plot of 1v vs 1[S] gives a line Slope = Krsquo

mVm

Intercept = 1Vm

][

111

][1

SV

K

Vv

SK

Vv

m

m

m

m

m

Substrate Inhibition

At high substrate concentrations Krsquom[S]ltlt1 and

inhibition is dominant

Plot of 1v vs [S] gives a straight line Slope = 1KS1 middot Vm

Intercept = 1Vm

mSm

S

m

VK

S

Vv

KS

Vv

1

1

][11

][1

1

max][

0][

SmKKS

Sddv

1V Igt0

I=0

1Vm

-1Km -1Kmapp 1[S]

1VIgt0

I=0

1Vm

-1Km-1Kmapp 1[S]

1Vmapp

1VIgt0

I=0

1Vm

-1Km 1[S]

1Vmapp

1V

1Vm

-1Km 1[S]

Competitive Uncompetitive

Non-Competitive Substrate Inhibition

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] onlyand competes with [S]increasing [S] overcomesInhibition by [II]

[II] binds to free [E] or [ES] complex Increasing [S] cannot overcome [II] inhibition

[II] binds to [ES] complex only increasing [S] favorsthe inhibition by [II]

E + S rarr ES rarr E + P + IIdarrEII

larr

uarr

E + S rarr ES rarr E + P + + II IIdarr darrEII + S rarrEIIS

larr

uarr uarr

E + S rarr ES rarr E + P + II darr EIIS

larr

uarr

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Kmrsquo [S] mM

vo

[S] mM

vo

II II

Km [S] mM

Vmax

II

Kmrsquo

VmaxrsquoVmaxrsquo

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax amp Km decreased

II

1[S]1Km

1vo

1 Vmax

II

Two parallellines

II

Intersect at X axis

1vo

1 Vmax

1[S]1Km 1[S]1Km

1 Vmax

1vo

Intersect at Y axis

= Kmrsquo

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

Enzymes

1048698 Cofactors

1048698 Coenzymes

1048698 Holoenzyme

1048698 Apoenzyme

bull Body conditions(temperature pressure etc) not good for reaction

bull Only enzymes can catalyse the reactions in this conditions

bull A special environment inside enzymes for reaction ACTIVE SITE

bull Molecule binds active site SUBSTRATE

How Enzymes Work

Enzymes Lower a Reactionrsquos Activation Energy

Each reaction has a transition state where thesubstrate is in an unstable short-livedchemicalstructural state

Free Energy of Activationis symbolized by ΔGDagger

Enzymes actby lowering the freeenergy of the transitionstate

Enzymes speed up metabolicreactions by lowering energy barriers

Enzyme speed reactions by lowering EAndash The transition state can be reached at moderate temperatures

Enzymes do not change delta Gndash It speed-up reactions that would occur eventually

Because enzymes are so selective they determine which chemical processes will occur at any time

Enzymes lower the free energy of activation by binding the transition state of the reaction better than the substrate

The enzyme must bind the substrate in the correct orientation otherwise there would be no reaction

Not a lock amp key but induced fit ndash the enzyme andor the substrate distort towards the transition state

Induced FitInduced Fit

A change in the shapeshape of an enzymersquos active site

Induced Induced by the substrate

Lock and Key Model

An enzyme binds a substrate in a region called the active site

Only certain substrates can fit the active site

Amino acid R groups in the active site help substrate bind

Enzyme-substrate complex forms

Substrate reacts to form product

Product is released

Enzyme Kinetics

- Kinetics The study of the rate of change

- Enzyme Kinetics Rate of chemical

reactions mediated by enzymes Enzymes can increase reaction rate by favoring or

enabling a different reaction pathway with

a lower activation energy making it easier

for the reaction to occur

Michaelis-Menten kinetics

V0 varies with [S]Vmax approachedasymptoticallyV0 is moles of

productformed per sec when [P]is low (close to zero time)E + SESE + P

Michaelis-Menten Model

V0 = Vmax x[S]([S] + Km)

Michaelis-Menten Equation

Determining initial velocity (when [P] is low)

Steady-state amp pre-steady-state conditions

At equilibrium no net change of [S] amp [P]or of [ES] amp [E]

At pre-steady-state[P] is low (close to zero time) hence V0 for initial

reaction velocity

At pre-steady state we can ignore the back reactions

Michaelis-Menten kinetics (summary)

Enzyme kinetics (Michaelis-Menten Graph) At fixed concentration of enzyme V0 is almost linearly proportional to

[S] when [S] is small but is nearly independent of [S] when [S] is large

Proposed Model E + S ES E + P

ES complex is a necessary intermediateObjective find an expression that relates rate of catalysis to theconcentrations of S amp E and the rates of individual steps

Start with V0 = k2[ES] and derive V0 = Vmax x[S]([S] + Km) This equation accounts for graph dataAt low [S] ([S] lt Km) V0 = (VmaxKm)[S]At high [S] ([S] gt Km) V0 = Vmax

When [S] = Km V0 = Vmax2 Thus Km = substrate concentration at which the reaction rate (V0) is half max

Range of Km values

Km provides approximation of [S] in vivo for many enzymes

Lineweaver-Burk plot (double-reciprocal)

Eadie-Hofstee plot

Hanes-Woolf Plot

Allosteric enzymes

Allosteric enzymes tend to be

multi-sub unit proteins

The reversible binding of an

allosteric modulator (here a

positive modulator M) affects

the substrate binding site

T

T

R

T

[S]

vo

Mechanism and Example of Allosteric Effect

S S

R

R

SS

RS

A

I

T[S]

vo

[S]

vo

(+)

(-) X X

X

R = Relax(active)

T = Tense(inactive)

Allosteric siteHomotropic(+)Concerted

Heterotropic(+)Sequential

Heterotropic(-)Concerted

Allosteric site

Kinetics Cooperation Models

(-)

(+)

(+)

Enzyme Inhibitors

bull Specific enzyme inhibitors regulate enzyme activity and help us

understand mechanism of enzyme action (Denaturing agents are not

inhibitors)

bull Irreversible inhibitors form covalent or very tight permanent bonds with

aa at the active site of the enzyme and render it inactive 3 classes

groupspecific reagents substrate analogs suicide inhibitors

bull Reversible inhibitors form an EI complex that can be dissociated back

to enzyme and free inhibitor 3 groups based on their mechanism of

action competitive non-competitive and uncompetitive

Enzyme Inhibition

Competitive inhibitors

bull Compete with substrate for binding to enzyme

bull E + S = ES or E + I = EI Both S and I cannot bind enzyme at the same time

bull In presence of I the equilibrium of E + S = ES is shifted to the left causing dissociation of ES

bull This can be reversed corrected by increasing [S]

bull Vmax is not changed KM is increased by (1 + IKi)

bull Eg AZT antibacterial sulfonamides the anticancer agent methotrexate etc

Competitive Inhibition

Kinetics of competitive inhibitor

Increase [S] toovercomeinhibition

Vmax attainable

Km is increased

Ki =dissociationconstant forinhibitor

V max unaltered Km increased

Non-competitive Inhibitors

bull Inhibitor binding site is distinct from substrate binding site Can bind to free enzyme E and to ES

bull E + I = EI ES + I = ESI or EI + S = ESI

bull Both EI and ESI are enzymatically inactive

bull The effective functional [E] (and [S]) is reduced

bull Reaction of unaffected ES proceeds normally

bull Inhibition cannot be reversed by increasing [S]

bull KM is not changed Vmax is decreased by (1 + IKi)

Mixed (Noncompetitive) Inhibition

Kinetics of non-competitive inhibitor

Increasing [S] cannotovercome inhibition

Less E availableV max is lowerKm remains the samefor available E

Km unaltered V max decreased

Uncompetitive Inhibitors

bull The inhibitor cannot bind to the enzyme directly but can only bind to the enzyme-substrate complex

bull ES + I = ESI

bull Both Vmax and KM are decreased by (1+IKi)

Uncompetitive Inhibition

Substrate Inhibition

Caused by high substrate concentrations

E + S ES E + PKm

rsquo k2

KS1

+

S

ES21

2

2

][][

][

][

]][[

][

]][[

Sm

m

mSi

KS

SK

SVv

ES

ESK

ES

ESSK

Substrate Inhibition

At low substrate concentrations [S]2Ks1ltlt1 and inhibition is not observed

Plot of 1v vs 1[S] gives a line Slope = Krsquo

mVm

Intercept = 1Vm

][

111

][1

SV

K

Vv

SK

Vv

m

m

m

m

m

Substrate Inhibition

At high substrate concentrations Krsquom[S]ltlt1 and

inhibition is dominant

Plot of 1v vs [S] gives a straight line Slope = 1KS1 middot Vm

Intercept = 1Vm

mSm

S

m

VK

S

Vv

KS

Vv

1

1

][11

][1

1

max][

0][

SmKKS

Sddv

1V Igt0

I=0

1Vm

-1Km -1Kmapp 1[S]

1VIgt0

I=0

1Vm

-1Km-1Kmapp 1[S]

1Vmapp

1VIgt0

I=0

1Vm

-1Km 1[S]

1Vmapp

1V

1Vm

-1Km 1[S]

Competitive Uncompetitive

Non-Competitive Substrate Inhibition

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] onlyand competes with [S]increasing [S] overcomesInhibition by [II]

[II] binds to free [E] or [ES] complex Increasing [S] cannot overcome [II] inhibition

[II] binds to [ES] complex only increasing [S] favorsthe inhibition by [II]

E + S rarr ES rarr E + P + IIdarrEII

larr

uarr

E + S rarr ES rarr E + P + + II IIdarr darrEII + S rarrEIIS

larr

uarr uarr

E + S rarr ES rarr E + P + II darr EIIS

larr

uarr

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Kmrsquo [S] mM

vo

[S] mM

vo

II II

Km [S] mM

Vmax

II

Kmrsquo

VmaxrsquoVmaxrsquo

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax amp Km decreased

II

1[S]1Km

1vo

1 Vmax

II

Two parallellines

II

Intersect at X axis

1vo

1 Vmax

1[S]1Km 1[S]1Km

1 Vmax

1vo

Intersect at Y axis

= Kmrsquo

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

bull Body conditions(temperature pressure etc) not good for reaction

bull Only enzymes can catalyse the reactions in this conditions

bull A special environment inside enzymes for reaction ACTIVE SITE

bull Molecule binds active site SUBSTRATE

How Enzymes Work

Enzymes Lower a Reactionrsquos Activation Energy

Each reaction has a transition state where thesubstrate is in an unstable short-livedchemicalstructural state

Free Energy of Activationis symbolized by ΔGDagger

Enzymes actby lowering the freeenergy of the transitionstate

Enzymes speed up metabolicreactions by lowering energy barriers

Enzyme speed reactions by lowering EAndash The transition state can be reached at moderate temperatures

Enzymes do not change delta Gndash It speed-up reactions that would occur eventually

Because enzymes are so selective they determine which chemical processes will occur at any time

Enzymes lower the free energy of activation by binding the transition state of the reaction better than the substrate

The enzyme must bind the substrate in the correct orientation otherwise there would be no reaction

Not a lock amp key but induced fit ndash the enzyme andor the substrate distort towards the transition state

Induced FitInduced Fit

A change in the shapeshape of an enzymersquos active site

Induced Induced by the substrate

Lock and Key Model

An enzyme binds a substrate in a region called the active site

Only certain substrates can fit the active site

Amino acid R groups in the active site help substrate bind

Enzyme-substrate complex forms

Substrate reacts to form product

Product is released

Enzyme Kinetics

- Kinetics The study of the rate of change

- Enzyme Kinetics Rate of chemical

reactions mediated by enzymes Enzymes can increase reaction rate by favoring or

enabling a different reaction pathway with

a lower activation energy making it easier

for the reaction to occur

Michaelis-Menten kinetics

V0 varies with [S]Vmax approachedasymptoticallyV0 is moles of

productformed per sec when [P]is low (close to zero time)E + SESE + P

Michaelis-Menten Model

V0 = Vmax x[S]([S] + Km)

Michaelis-Menten Equation

Determining initial velocity (when [P] is low)

Steady-state amp pre-steady-state conditions

At equilibrium no net change of [S] amp [P]or of [ES] amp [E]

At pre-steady-state[P] is low (close to zero time) hence V0 for initial

reaction velocity

At pre-steady state we can ignore the back reactions

Michaelis-Menten kinetics (summary)

Enzyme kinetics (Michaelis-Menten Graph) At fixed concentration of enzyme V0 is almost linearly proportional to

[S] when [S] is small but is nearly independent of [S] when [S] is large

Proposed Model E + S ES E + P

ES complex is a necessary intermediateObjective find an expression that relates rate of catalysis to theconcentrations of S amp E and the rates of individual steps

Start with V0 = k2[ES] and derive V0 = Vmax x[S]([S] + Km) This equation accounts for graph dataAt low [S] ([S] lt Km) V0 = (VmaxKm)[S]At high [S] ([S] gt Km) V0 = Vmax

When [S] = Km V0 = Vmax2 Thus Km = substrate concentration at which the reaction rate (V0) is half max

Range of Km values

Km provides approximation of [S] in vivo for many enzymes

Lineweaver-Burk plot (double-reciprocal)

Eadie-Hofstee plot

Hanes-Woolf Plot

Allosteric enzymes

Allosteric enzymes tend to be

multi-sub unit proteins

The reversible binding of an

allosteric modulator (here a

positive modulator M) affects

the substrate binding site

T

T

R

T

[S]

vo

Mechanism and Example of Allosteric Effect

S S

R

R

SS

RS

A

I

T[S]

vo

[S]

vo

(+)

(-) X X

X

R = Relax(active)

T = Tense(inactive)

Allosteric siteHomotropic(+)Concerted

Heterotropic(+)Sequential

Heterotropic(-)Concerted

Allosteric site

Kinetics Cooperation Models

(-)

(+)

(+)

Enzyme Inhibitors

bull Specific enzyme inhibitors regulate enzyme activity and help us

understand mechanism of enzyme action (Denaturing agents are not

inhibitors)

bull Irreversible inhibitors form covalent or very tight permanent bonds with

aa at the active site of the enzyme and render it inactive 3 classes

groupspecific reagents substrate analogs suicide inhibitors

bull Reversible inhibitors form an EI complex that can be dissociated back

to enzyme and free inhibitor 3 groups based on their mechanism of

action competitive non-competitive and uncompetitive

Enzyme Inhibition

Competitive inhibitors

bull Compete with substrate for binding to enzyme

bull E + S = ES or E + I = EI Both S and I cannot bind enzyme at the same time

bull In presence of I the equilibrium of E + S = ES is shifted to the left causing dissociation of ES

bull This can be reversed corrected by increasing [S]

bull Vmax is not changed KM is increased by (1 + IKi)

bull Eg AZT antibacterial sulfonamides the anticancer agent methotrexate etc

Competitive Inhibition

Kinetics of competitive inhibitor

Increase [S] toovercomeinhibition

Vmax attainable

Km is increased

Ki =dissociationconstant forinhibitor

V max unaltered Km increased

Non-competitive Inhibitors

bull Inhibitor binding site is distinct from substrate binding site Can bind to free enzyme E and to ES

bull E + I = EI ES + I = ESI or EI + S = ESI

bull Both EI and ESI are enzymatically inactive

bull The effective functional [E] (and [S]) is reduced

bull Reaction of unaffected ES proceeds normally

bull Inhibition cannot be reversed by increasing [S]

bull KM is not changed Vmax is decreased by (1 + IKi)

Mixed (Noncompetitive) Inhibition

Kinetics of non-competitive inhibitor

Increasing [S] cannotovercome inhibition

Less E availableV max is lowerKm remains the samefor available E

Km unaltered V max decreased

Uncompetitive Inhibitors

bull The inhibitor cannot bind to the enzyme directly but can only bind to the enzyme-substrate complex

bull ES + I = ESI

bull Both Vmax and KM are decreased by (1+IKi)

Uncompetitive Inhibition

Substrate Inhibition

Caused by high substrate concentrations

E + S ES E + PKm

rsquo k2

KS1

+

S

ES21

2

2

][][

][

][

]][[

][

]][[

Sm

m

mSi

KS

SK

SVv

ES

ESK

ES

ESSK

Substrate Inhibition

At low substrate concentrations [S]2Ks1ltlt1 and inhibition is not observed

Plot of 1v vs 1[S] gives a line Slope = Krsquo

mVm

Intercept = 1Vm

][

111

][1

SV

K

Vv

SK

Vv

m

m

m

m

m

Substrate Inhibition

At high substrate concentrations Krsquom[S]ltlt1 and

inhibition is dominant

Plot of 1v vs [S] gives a straight line Slope = 1KS1 middot Vm

Intercept = 1Vm

mSm

S

m

VK

S

Vv

KS

Vv

1

1

][11

][1

1

max][

0][

SmKKS

Sddv

1V Igt0

I=0

1Vm

-1Km -1Kmapp 1[S]

1VIgt0

I=0

1Vm

-1Km-1Kmapp 1[S]

1Vmapp

1VIgt0

I=0

1Vm

-1Km 1[S]

1Vmapp

1V

1Vm

-1Km 1[S]

Competitive Uncompetitive

Non-Competitive Substrate Inhibition

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] onlyand competes with [S]increasing [S] overcomesInhibition by [II]

[II] binds to free [E] or [ES] complex Increasing [S] cannot overcome [II] inhibition

[II] binds to [ES] complex only increasing [S] favorsthe inhibition by [II]

E + S rarr ES rarr E + P + IIdarrEII

larr

uarr

E + S rarr ES rarr E + P + + II IIdarr darrEII + S rarrEIIS

larr

uarr uarr

E + S rarr ES rarr E + P + II darr EIIS

larr

uarr

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Kmrsquo [S] mM

vo

[S] mM

vo

II II

Km [S] mM

Vmax

II

Kmrsquo

VmaxrsquoVmaxrsquo

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax amp Km decreased

II

1[S]1Km

1vo

1 Vmax

II

Two parallellines

II

Intersect at X axis

1vo

1 Vmax

1[S]1Km 1[S]1Km

1 Vmax

1vo

Intersect at Y axis

= Kmrsquo

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

Enzymes Lower a Reactionrsquos Activation Energy

Each reaction has a transition state where thesubstrate is in an unstable short-livedchemicalstructural state

Free Energy of Activationis symbolized by ΔGDagger

Enzymes actby lowering the freeenergy of the transitionstate

Enzymes speed up metabolicreactions by lowering energy barriers

Enzyme speed reactions by lowering EAndash The transition state can be reached at moderate temperatures

Enzymes do not change delta Gndash It speed-up reactions that would occur eventually

Because enzymes are so selective they determine which chemical processes will occur at any time

Enzymes lower the free energy of activation by binding the transition state of the reaction better than the substrate

The enzyme must bind the substrate in the correct orientation otherwise there would be no reaction

Not a lock amp key but induced fit ndash the enzyme andor the substrate distort towards the transition state

Induced FitInduced Fit

A change in the shapeshape of an enzymersquos active site

Induced Induced by the substrate

Lock and Key Model

An enzyme binds a substrate in a region called the active site

Only certain substrates can fit the active site

Amino acid R groups in the active site help substrate bind

Enzyme-substrate complex forms

Substrate reacts to form product

Product is released

Enzyme Kinetics

- Kinetics The study of the rate of change

- Enzyme Kinetics Rate of chemical

reactions mediated by enzymes Enzymes can increase reaction rate by favoring or

enabling a different reaction pathway with

a lower activation energy making it easier

for the reaction to occur

Michaelis-Menten kinetics

V0 varies with [S]Vmax approachedasymptoticallyV0 is moles of

productformed per sec when [P]is low (close to zero time)E + SESE + P

Michaelis-Menten Model

V0 = Vmax x[S]([S] + Km)

Michaelis-Menten Equation

Determining initial velocity (when [P] is low)

Steady-state amp pre-steady-state conditions

At equilibrium no net change of [S] amp [P]or of [ES] amp [E]

At pre-steady-state[P] is low (close to zero time) hence V0 for initial

reaction velocity

At pre-steady state we can ignore the back reactions

Michaelis-Menten kinetics (summary)

Enzyme kinetics (Michaelis-Menten Graph) At fixed concentration of enzyme V0 is almost linearly proportional to

[S] when [S] is small but is nearly independent of [S] when [S] is large

Proposed Model E + S ES E + P

ES complex is a necessary intermediateObjective find an expression that relates rate of catalysis to theconcentrations of S amp E and the rates of individual steps

Start with V0 = k2[ES] and derive V0 = Vmax x[S]([S] + Km) This equation accounts for graph dataAt low [S] ([S] lt Km) V0 = (VmaxKm)[S]At high [S] ([S] gt Km) V0 = Vmax

When [S] = Km V0 = Vmax2 Thus Km = substrate concentration at which the reaction rate (V0) is half max

Range of Km values

Km provides approximation of [S] in vivo for many enzymes

Lineweaver-Burk plot (double-reciprocal)

Eadie-Hofstee plot

Hanes-Woolf Plot

Allosteric enzymes

Allosteric enzymes tend to be

multi-sub unit proteins

The reversible binding of an

allosteric modulator (here a

positive modulator M) affects

the substrate binding site

T

T

R

T

[S]

vo

Mechanism and Example of Allosteric Effect

S S

R

R

SS

RS

A

I

T[S]

vo

[S]

vo

(+)

(-) X X

X

R = Relax(active)

T = Tense(inactive)

Allosteric siteHomotropic(+)Concerted

Heterotropic(+)Sequential

Heterotropic(-)Concerted

Allosteric site

Kinetics Cooperation Models

(-)

(+)

(+)

Enzyme Inhibitors

bull Specific enzyme inhibitors regulate enzyme activity and help us

understand mechanism of enzyme action (Denaturing agents are not

inhibitors)

bull Irreversible inhibitors form covalent or very tight permanent bonds with

aa at the active site of the enzyme and render it inactive 3 classes

groupspecific reagents substrate analogs suicide inhibitors

bull Reversible inhibitors form an EI complex that can be dissociated back

to enzyme and free inhibitor 3 groups based on their mechanism of

action competitive non-competitive and uncompetitive

Enzyme Inhibition

Competitive inhibitors

bull Compete with substrate for binding to enzyme

bull E + S = ES or E + I = EI Both S and I cannot bind enzyme at the same time

bull In presence of I the equilibrium of E + S = ES is shifted to the left causing dissociation of ES

bull This can be reversed corrected by increasing [S]

bull Vmax is not changed KM is increased by (1 + IKi)

bull Eg AZT antibacterial sulfonamides the anticancer agent methotrexate etc

Competitive Inhibition

Kinetics of competitive inhibitor

Increase [S] toovercomeinhibition

Vmax attainable

Km is increased

Ki =dissociationconstant forinhibitor

V max unaltered Km increased

Non-competitive Inhibitors

bull Inhibitor binding site is distinct from substrate binding site Can bind to free enzyme E and to ES

bull E + I = EI ES + I = ESI or EI + S = ESI

bull Both EI and ESI are enzymatically inactive

bull The effective functional [E] (and [S]) is reduced

bull Reaction of unaffected ES proceeds normally

bull Inhibition cannot be reversed by increasing [S]

bull KM is not changed Vmax is decreased by (1 + IKi)

Mixed (Noncompetitive) Inhibition

Kinetics of non-competitive inhibitor

Increasing [S] cannotovercome inhibition

Less E availableV max is lowerKm remains the samefor available E

Km unaltered V max decreased

Uncompetitive Inhibitors

bull The inhibitor cannot bind to the enzyme directly but can only bind to the enzyme-substrate complex

bull ES + I = ESI

bull Both Vmax and KM are decreased by (1+IKi)

Uncompetitive Inhibition

Substrate Inhibition

Caused by high substrate concentrations

E + S ES E + PKm

rsquo k2

KS1

+

S

ES21

2

2

][][

][

][

]][[

][

]][[

Sm

m

mSi

KS

SK

SVv

ES

ESK

ES

ESSK

Substrate Inhibition

At low substrate concentrations [S]2Ks1ltlt1 and inhibition is not observed

Plot of 1v vs 1[S] gives a line Slope = Krsquo

mVm

Intercept = 1Vm

][

111

][1

SV

K

Vv

SK

Vv

m

m

m

m

m

Substrate Inhibition

At high substrate concentrations Krsquom[S]ltlt1 and

inhibition is dominant

Plot of 1v vs [S] gives a straight line Slope = 1KS1 middot Vm

Intercept = 1Vm

mSm

S

m

VK

S

Vv

KS

Vv

1

1

][11

][1

1

max][

0][

SmKKS

Sddv

1V Igt0

I=0

1Vm

-1Km -1Kmapp 1[S]

1VIgt0

I=0

1Vm

-1Km-1Kmapp 1[S]

1Vmapp

1VIgt0

I=0

1Vm

-1Km 1[S]

1Vmapp

1V

1Vm

-1Km 1[S]

Competitive Uncompetitive

Non-Competitive Substrate Inhibition

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] onlyand competes with [S]increasing [S] overcomesInhibition by [II]

[II] binds to free [E] or [ES] complex Increasing [S] cannot overcome [II] inhibition

[II] binds to [ES] complex only increasing [S] favorsthe inhibition by [II]

E + S rarr ES rarr E + P + IIdarrEII

larr

uarr

E + S rarr ES rarr E + P + + II IIdarr darrEII + S rarrEIIS

larr

uarr uarr

E + S rarr ES rarr E + P + II darr EIIS

larr

uarr

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Kmrsquo [S] mM

vo

[S] mM

vo

II II

Km [S] mM

Vmax

II

Kmrsquo

VmaxrsquoVmaxrsquo

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax amp Km decreased

II

1[S]1Km

1vo

1 Vmax

II

Two parallellines

II

Intersect at X axis

1vo

1 Vmax

1[S]1Km 1[S]1Km

1 Vmax

1vo

Intersect at Y axis

= Kmrsquo

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

Each reaction has a transition state where thesubstrate is in an unstable short-livedchemicalstructural state

Free Energy of Activationis symbolized by ΔGDagger

Enzymes actby lowering the freeenergy of the transitionstate

Enzymes speed up metabolicreactions by lowering energy barriers

Enzyme speed reactions by lowering EAndash The transition state can be reached at moderate temperatures

Enzymes do not change delta Gndash It speed-up reactions that would occur eventually

Because enzymes are so selective they determine which chemical processes will occur at any time

Enzymes lower the free energy of activation by binding the transition state of the reaction better than the substrate

The enzyme must bind the substrate in the correct orientation otherwise there would be no reaction

Not a lock amp key but induced fit ndash the enzyme andor the substrate distort towards the transition state

Induced FitInduced Fit

A change in the shapeshape of an enzymersquos active site

Induced Induced by the substrate

Lock and Key Model

An enzyme binds a substrate in a region called the active site

Only certain substrates can fit the active site

Amino acid R groups in the active site help substrate bind

Enzyme-substrate complex forms

Substrate reacts to form product

Product is released

Enzyme Kinetics

- Kinetics The study of the rate of change

- Enzyme Kinetics Rate of chemical

reactions mediated by enzymes Enzymes can increase reaction rate by favoring or

enabling a different reaction pathway with

a lower activation energy making it easier

for the reaction to occur

Michaelis-Menten kinetics

V0 varies with [S]Vmax approachedasymptoticallyV0 is moles of

productformed per sec when [P]is low (close to zero time)E + SESE + P

Michaelis-Menten Model

V0 = Vmax x[S]([S] + Km)

Michaelis-Menten Equation

Determining initial velocity (when [P] is low)

Steady-state amp pre-steady-state conditions

At equilibrium no net change of [S] amp [P]or of [ES] amp [E]

At pre-steady-state[P] is low (close to zero time) hence V0 for initial

reaction velocity

At pre-steady state we can ignore the back reactions

Michaelis-Menten kinetics (summary)

Enzyme kinetics (Michaelis-Menten Graph) At fixed concentration of enzyme V0 is almost linearly proportional to

[S] when [S] is small but is nearly independent of [S] when [S] is large

Proposed Model E + S ES E + P

ES complex is a necessary intermediateObjective find an expression that relates rate of catalysis to theconcentrations of S amp E and the rates of individual steps

Start with V0 = k2[ES] and derive V0 = Vmax x[S]([S] + Km) This equation accounts for graph dataAt low [S] ([S] lt Km) V0 = (VmaxKm)[S]At high [S] ([S] gt Km) V0 = Vmax

When [S] = Km V0 = Vmax2 Thus Km = substrate concentration at which the reaction rate (V0) is half max

Range of Km values

Km provides approximation of [S] in vivo for many enzymes

Lineweaver-Burk plot (double-reciprocal)

Eadie-Hofstee plot

Hanes-Woolf Plot

Allosteric enzymes

Allosteric enzymes tend to be

multi-sub unit proteins

The reversible binding of an

allosteric modulator (here a

positive modulator M) affects

the substrate binding site

T

T

R

T

[S]

vo

Mechanism and Example of Allosteric Effect

S S

R

R

SS

RS

A

I

T[S]

vo

[S]

vo

(+)

(-) X X

X

R = Relax(active)

T = Tense(inactive)

Allosteric siteHomotropic(+)Concerted

Heterotropic(+)Sequential

Heterotropic(-)Concerted

Allosteric site

Kinetics Cooperation Models

(-)

(+)

(+)

Enzyme Inhibitors

bull Specific enzyme inhibitors regulate enzyme activity and help us

understand mechanism of enzyme action (Denaturing agents are not

inhibitors)

bull Irreversible inhibitors form covalent or very tight permanent bonds with

aa at the active site of the enzyme and render it inactive 3 classes

groupspecific reagents substrate analogs suicide inhibitors

bull Reversible inhibitors form an EI complex that can be dissociated back

to enzyme and free inhibitor 3 groups based on their mechanism of

action competitive non-competitive and uncompetitive

Enzyme Inhibition

Competitive inhibitors

bull Compete with substrate for binding to enzyme

bull E + S = ES or E + I = EI Both S and I cannot bind enzyme at the same time

bull In presence of I the equilibrium of E + S = ES is shifted to the left causing dissociation of ES

bull This can be reversed corrected by increasing [S]

bull Vmax is not changed KM is increased by (1 + IKi)

bull Eg AZT antibacterial sulfonamides the anticancer agent methotrexate etc

Competitive Inhibition

Kinetics of competitive inhibitor

Increase [S] toovercomeinhibition

Vmax attainable

Km is increased

Ki =dissociationconstant forinhibitor

V max unaltered Km increased

Non-competitive Inhibitors

bull Inhibitor binding site is distinct from substrate binding site Can bind to free enzyme E and to ES

bull E + I = EI ES + I = ESI or EI + S = ESI

bull Both EI and ESI are enzymatically inactive

bull The effective functional [E] (and [S]) is reduced

bull Reaction of unaffected ES proceeds normally

bull Inhibition cannot be reversed by increasing [S]

bull KM is not changed Vmax is decreased by (1 + IKi)

Mixed (Noncompetitive) Inhibition

Kinetics of non-competitive inhibitor

Increasing [S] cannotovercome inhibition

Less E availableV max is lowerKm remains the samefor available E

Km unaltered V max decreased

Uncompetitive Inhibitors

bull The inhibitor cannot bind to the enzyme directly but can only bind to the enzyme-substrate complex

bull ES + I = ESI

bull Both Vmax and KM are decreased by (1+IKi)

Uncompetitive Inhibition

Substrate Inhibition

Caused by high substrate concentrations

E + S ES E + PKm

rsquo k2

KS1

+

S

ES21

2

2

][][

][

][

]][[

][

]][[

Sm

m

mSi

KS

SK

SVv

ES

ESK

ES

ESSK

Substrate Inhibition

At low substrate concentrations [S]2Ks1ltlt1 and inhibition is not observed

Plot of 1v vs 1[S] gives a line Slope = Krsquo

mVm

Intercept = 1Vm

][

111

][1

SV

K

Vv

SK

Vv

m

m

m

m

m

Substrate Inhibition

At high substrate concentrations Krsquom[S]ltlt1 and

inhibition is dominant

Plot of 1v vs [S] gives a straight line Slope = 1KS1 middot Vm

Intercept = 1Vm

mSm

S

m

VK

S

Vv

KS

Vv

1

1

][11

][1

1

max][

0][

SmKKS

Sddv

1V Igt0

I=0

1Vm

-1Km -1Kmapp 1[S]

1VIgt0

I=0

1Vm

-1Km-1Kmapp 1[S]

1Vmapp

1VIgt0

I=0

1Vm

-1Km 1[S]

1Vmapp

1V

1Vm

-1Km 1[S]

Competitive Uncompetitive

Non-Competitive Substrate Inhibition

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] onlyand competes with [S]increasing [S] overcomesInhibition by [II]

[II] binds to free [E] or [ES] complex Increasing [S] cannot overcome [II] inhibition

[II] binds to [ES] complex only increasing [S] favorsthe inhibition by [II]

E + S rarr ES rarr E + P + IIdarrEII

larr

uarr

E + S rarr ES rarr E + P + + II IIdarr darrEII + S rarrEIIS

larr

uarr uarr

E + S rarr ES rarr E + P + II darr EIIS

larr

uarr

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Kmrsquo [S] mM

vo

[S] mM

vo

II II

Km [S] mM

Vmax

II

Kmrsquo

VmaxrsquoVmaxrsquo

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax amp Km decreased

II

1[S]1Km

1vo

1 Vmax

II

Two parallellines

II

Intersect at X axis

1vo

1 Vmax

1[S]1Km 1[S]1Km

1 Vmax

1vo

Intersect at Y axis

= Kmrsquo

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

Enzymes speed up metabolicreactions by lowering energy barriers

Enzyme speed reactions by lowering EAndash The transition state can be reached at moderate temperatures

Enzymes do not change delta Gndash It speed-up reactions that would occur eventually

Because enzymes are so selective they determine which chemical processes will occur at any time

Enzymes lower the free energy of activation by binding the transition state of the reaction better than the substrate

The enzyme must bind the substrate in the correct orientation otherwise there would be no reaction

Not a lock amp key but induced fit ndash the enzyme andor the substrate distort towards the transition state

Induced FitInduced Fit

A change in the shapeshape of an enzymersquos active site

Induced Induced by the substrate

Lock and Key Model

An enzyme binds a substrate in a region called the active site

Only certain substrates can fit the active site

Amino acid R groups in the active site help substrate bind

Enzyme-substrate complex forms

Substrate reacts to form product

Product is released

Enzyme Kinetics

- Kinetics The study of the rate of change

- Enzyme Kinetics Rate of chemical

reactions mediated by enzymes Enzymes can increase reaction rate by favoring or

enabling a different reaction pathway with

a lower activation energy making it easier

for the reaction to occur

Michaelis-Menten kinetics

V0 varies with [S]Vmax approachedasymptoticallyV0 is moles of

productformed per sec when [P]is low (close to zero time)E + SESE + P

Michaelis-Menten Model

V0 = Vmax x[S]([S] + Km)

Michaelis-Menten Equation

Determining initial velocity (when [P] is low)

Steady-state amp pre-steady-state conditions

At equilibrium no net change of [S] amp [P]or of [ES] amp [E]

At pre-steady-state[P] is low (close to zero time) hence V0 for initial

reaction velocity

At pre-steady state we can ignore the back reactions

Michaelis-Menten kinetics (summary)

Enzyme kinetics (Michaelis-Menten Graph) At fixed concentration of enzyme V0 is almost linearly proportional to

[S] when [S] is small but is nearly independent of [S] when [S] is large

Proposed Model E + S ES E + P

ES complex is a necessary intermediateObjective find an expression that relates rate of catalysis to theconcentrations of S amp E and the rates of individual steps

Start with V0 = k2[ES] and derive V0 = Vmax x[S]([S] + Km) This equation accounts for graph dataAt low [S] ([S] lt Km) V0 = (VmaxKm)[S]At high [S] ([S] gt Km) V0 = Vmax

When [S] = Km V0 = Vmax2 Thus Km = substrate concentration at which the reaction rate (V0) is half max

Range of Km values

Km provides approximation of [S] in vivo for many enzymes

Lineweaver-Burk plot (double-reciprocal)

Eadie-Hofstee plot

Hanes-Woolf Plot

Allosteric enzymes

Allosteric enzymes tend to be

multi-sub unit proteins

The reversible binding of an

allosteric modulator (here a

positive modulator M) affects

the substrate binding site

T

T

R

T

[S]

vo

Mechanism and Example of Allosteric Effect

S S

R

R

SS

RS

A

I

T[S]

vo

[S]

vo

(+)

(-) X X

X

R = Relax(active)

T = Tense(inactive)

Allosteric siteHomotropic(+)Concerted

Heterotropic(+)Sequential

Heterotropic(-)Concerted

Allosteric site

Kinetics Cooperation Models

(-)

(+)

(+)

Enzyme Inhibitors

bull Specific enzyme inhibitors regulate enzyme activity and help us

understand mechanism of enzyme action (Denaturing agents are not

inhibitors)

bull Irreversible inhibitors form covalent or very tight permanent bonds with

aa at the active site of the enzyme and render it inactive 3 classes

groupspecific reagents substrate analogs suicide inhibitors

bull Reversible inhibitors form an EI complex that can be dissociated back

to enzyme and free inhibitor 3 groups based on their mechanism of

action competitive non-competitive and uncompetitive

Enzyme Inhibition

Competitive inhibitors

bull Compete with substrate for binding to enzyme

bull E + S = ES or E + I = EI Both S and I cannot bind enzyme at the same time

bull In presence of I the equilibrium of E + S = ES is shifted to the left causing dissociation of ES

bull This can be reversed corrected by increasing [S]

bull Vmax is not changed KM is increased by (1 + IKi)

bull Eg AZT antibacterial sulfonamides the anticancer agent methotrexate etc

Competitive Inhibition

Kinetics of competitive inhibitor

Increase [S] toovercomeinhibition

Vmax attainable

Km is increased

Ki =dissociationconstant forinhibitor

V max unaltered Km increased

Non-competitive Inhibitors

bull Inhibitor binding site is distinct from substrate binding site Can bind to free enzyme E and to ES

bull E + I = EI ES + I = ESI or EI + S = ESI

bull Both EI and ESI are enzymatically inactive

bull The effective functional [E] (and [S]) is reduced

bull Reaction of unaffected ES proceeds normally

bull Inhibition cannot be reversed by increasing [S]

bull KM is not changed Vmax is decreased by (1 + IKi)

Mixed (Noncompetitive) Inhibition

Kinetics of non-competitive inhibitor

Increasing [S] cannotovercome inhibition

Less E availableV max is lowerKm remains the samefor available E

Km unaltered V max decreased

Uncompetitive Inhibitors

bull The inhibitor cannot bind to the enzyme directly but can only bind to the enzyme-substrate complex

bull ES + I = ESI

bull Both Vmax and KM are decreased by (1+IKi)

Uncompetitive Inhibition

Substrate Inhibition

Caused by high substrate concentrations

E + S ES E + PKm

rsquo k2

KS1

+

S

ES21

2

2

][][

][

][

]][[

][

]][[

Sm

m

mSi

KS

SK

SVv

ES

ESK

ES

ESSK

Substrate Inhibition

At low substrate concentrations [S]2Ks1ltlt1 and inhibition is not observed

Plot of 1v vs 1[S] gives a line Slope = Krsquo

mVm

Intercept = 1Vm

][

111

][1

SV

K

Vv

SK

Vv

m

m

m

m

m

Substrate Inhibition

At high substrate concentrations Krsquom[S]ltlt1 and

inhibition is dominant

Plot of 1v vs [S] gives a straight line Slope = 1KS1 middot Vm

Intercept = 1Vm

mSm

S

m

VK

S

Vv

KS

Vv

1

1

][11

][1

1

max][

0][

SmKKS

Sddv

1V Igt0

I=0

1Vm

-1Km -1Kmapp 1[S]

1VIgt0

I=0

1Vm

-1Km-1Kmapp 1[S]

1Vmapp

1VIgt0

I=0

1Vm

-1Km 1[S]

1Vmapp

1V

1Vm

-1Km 1[S]

Competitive Uncompetitive

Non-Competitive Substrate Inhibition

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] onlyand competes with [S]increasing [S] overcomesInhibition by [II]

[II] binds to free [E] or [ES] complex Increasing [S] cannot overcome [II] inhibition

[II] binds to [ES] complex only increasing [S] favorsthe inhibition by [II]

E + S rarr ES rarr E + P + IIdarrEII

larr

uarr

E + S rarr ES rarr E + P + + II IIdarr darrEII + S rarrEIIS

larr

uarr uarr

E + S rarr ES rarr E + P + II darr EIIS

larr

uarr

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Kmrsquo [S] mM

vo

[S] mM

vo

II II

Km [S] mM

Vmax

II

Kmrsquo

VmaxrsquoVmaxrsquo

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax amp Km decreased

II

1[S]1Km

1vo

1 Vmax

II

Two parallellines

II

Intersect at X axis

1vo

1 Vmax

1[S]1Km 1[S]1Km

1 Vmax

1vo

Intersect at Y axis

= Kmrsquo

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

Enzymes lower the free energy of activation by binding the transition state of the reaction better than the substrate

The enzyme must bind the substrate in the correct orientation otherwise there would be no reaction

Not a lock amp key but induced fit ndash the enzyme andor the substrate distort towards the transition state

Induced FitInduced Fit

A change in the shapeshape of an enzymersquos active site

Induced Induced by the substrate

Lock and Key Model

An enzyme binds a substrate in a region called the active site

Only certain substrates can fit the active site

Amino acid R groups in the active site help substrate bind

Enzyme-substrate complex forms

Substrate reacts to form product

Product is released

Enzyme Kinetics

- Kinetics The study of the rate of change

- Enzyme Kinetics Rate of chemical

reactions mediated by enzymes Enzymes can increase reaction rate by favoring or

enabling a different reaction pathway with

a lower activation energy making it easier

for the reaction to occur

Michaelis-Menten kinetics

V0 varies with [S]Vmax approachedasymptoticallyV0 is moles of

productformed per sec when [P]is low (close to zero time)E + SESE + P

Michaelis-Menten Model

V0 = Vmax x[S]([S] + Km)

Michaelis-Menten Equation

Determining initial velocity (when [P] is low)

Steady-state amp pre-steady-state conditions

At equilibrium no net change of [S] amp [P]or of [ES] amp [E]

At pre-steady-state[P] is low (close to zero time) hence V0 for initial

reaction velocity

At pre-steady state we can ignore the back reactions

Michaelis-Menten kinetics (summary)

Enzyme kinetics (Michaelis-Menten Graph) At fixed concentration of enzyme V0 is almost linearly proportional to

[S] when [S] is small but is nearly independent of [S] when [S] is large

Proposed Model E + S ES E + P

ES complex is a necessary intermediateObjective find an expression that relates rate of catalysis to theconcentrations of S amp E and the rates of individual steps

Start with V0 = k2[ES] and derive V0 = Vmax x[S]([S] + Km) This equation accounts for graph dataAt low [S] ([S] lt Km) V0 = (VmaxKm)[S]At high [S] ([S] gt Km) V0 = Vmax

When [S] = Km V0 = Vmax2 Thus Km = substrate concentration at which the reaction rate (V0) is half max

Range of Km values

Km provides approximation of [S] in vivo for many enzymes

Lineweaver-Burk plot (double-reciprocal)

Eadie-Hofstee plot

Hanes-Woolf Plot

Allosteric enzymes

Allosteric enzymes tend to be

multi-sub unit proteins

The reversible binding of an

allosteric modulator (here a

positive modulator M) affects

the substrate binding site

T

T

R

T

[S]

vo

Mechanism and Example of Allosteric Effect

S S

R

R

SS

RS

A

I

T[S]

vo

[S]

vo

(+)

(-) X X

X

R = Relax(active)

T = Tense(inactive)

Allosteric siteHomotropic(+)Concerted

Heterotropic(+)Sequential

Heterotropic(-)Concerted

Allosteric site

Kinetics Cooperation Models

(-)

(+)

(+)

Enzyme Inhibitors

bull Specific enzyme inhibitors regulate enzyme activity and help us

understand mechanism of enzyme action (Denaturing agents are not

inhibitors)

bull Irreversible inhibitors form covalent or very tight permanent bonds with

aa at the active site of the enzyme and render it inactive 3 classes

groupspecific reagents substrate analogs suicide inhibitors

bull Reversible inhibitors form an EI complex that can be dissociated back

to enzyme and free inhibitor 3 groups based on their mechanism of

action competitive non-competitive and uncompetitive

Enzyme Inhibition

Competitive inhibitors

bull Compete with substrate for binding to enzyme

bull E + S = ES or E + I = EI Both S and I cannot bind enzyme at the same time

bull In presence of I the equilibrium of E + S = ES is shifted to the left causing dissociation of ES

bull This can be reversed corrected by increasing [S]

bull Vmax is not changed KM is increased by (1 + IKi)

bull Eg AZT antibacterial sulfonamides the anticancer agent methotrexate etc

Competitive Inhibition

Kinetics of competitive inhibitor

Increase [S] toovercomeinhibition

Vmax attainable

Km is increased

Ki =dissociationconstant forinhibitor

V max unaltered Km increased

Non-competitive Inhibitors

bull Inhibitor binding site is distinct from substrate binding site Can bind to free enzyme E and to ES

bull E + I = EI ES + I = ESI or EI + S = ESI

bull Both EI and ESI are enzymatically inactive

bull The effective functional [E] (and [S]) is reduced

bull Reaction of unaffected ES proceeds normally

bull Inhibition cannot be reversed by increasing [S]

bull KM is not changed Vmax is decreased by (1 + IKi)

Mixed (Noncompetitive) Inhibition

Kinetics of non-competitive inhibitor

Increasing [S] cannotovercome inhibition

Less E availableV max is lowerKm remains the samefor available E

Km unaltered V max decreased

Uncompetitive Inhibitors

bull The inhibitor cannot bind to the enzyme directly but can only bind to the enzyme-substrate complex

bull ES + I = ESI

bull Both Vmax and KM are decreased by (1+IKi)

Uncompetitive Inhibition

Substrate Inhibition

Caused by high substrate concentrations

E + S ES E + PKm

rsquo k2

KS1

+

S

ES21

2

2

][][

][

][

]][[

][

]][[

Sm

m

mSi

KS

SK

SVv

ES

ESK

ES

ESSK

Substrate Inhibition

At low substrate concentrations [S]2Ks1ltlt1 and inhibition is not observed

Plot of 1v vs 1[S] gives a line Slope = Krsquo

mVm

Intercept = 1Vm

][

111

][1

SV

K

Vv

SK

Vv

m

m

m

m

m

Substrate Inhibition

At high substrate concentrations Krsquom[S]ltlt1 and

inhibition is dominant

Plot of 1v vs [S] gives a straight line Slope = 1KS1 middot Vm

Intercept = 1Vm

mSm

S

m

VK

S

Vv

KS

Vv

1

1

][11

][1

1

max][

0][

SmKKS

Sddv

1V Igt0

I=0

1Vm

-1Km -1Kmapp 1[S]

1VIgt0

I=0

1Vm

-1Km-1Kmapp 1[S]

1Vmapp

1VIgt0

I=0

1Vm

-1Km 1[S]

1Vmapp

1V

1Vm

-1Km 1[S]

Competitive Uncompetitive

Non-Competitive Substrate Inhibition

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] onlyand competes with [S]increasing [S] overcomesInhibition by [II]

[II] binds to free [E] or [ES] complex Increasing [S] cannot overcome [II] inhibition

[II] binds to [ES] complex only increasing [S] favorsthe inhibition by [II]

E + S rarr ES rarr E + P + IIdarrEII

larr

uarr

E + S rarr ES rarr E + P + + II IIdarr darrEII + S rarrEIIS

larr

uarr uarr

E + S rarr ES rarr E + P + II darr EIIS

larr

uarr

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Kmrsquo [S] mM

vo

[S] mM

vo

II II

Km [S] mM

Vmax

II

Kmrsquo

VmaxrsquoVmaxrsquo

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax amp Km decreased

II

1[S]1Km

1vo

1 Vmax

II

Two parallellines

II

Intersect at X axis

1vo

1 Vmax

1[S]1Km 1[S]1Km

1 Vmax

1vo

Intersect at Y axis

= Kmrsquo

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

Induced FitInduced Fit

A change in the shapeshape of an enzymersquos active site

Induced Induced by the substrate

Lock and Key Model

An enzyme binds a substrate in a region called the active site

Only certain substrates can fit the active site

Amino acid R groups in the active site help substrate bind

Enzyme-substrate complex forms

Substrate reacts to form product

Product is released

Enzyme Kinetics

- Kinetics The study of the rate of change

- Enzyme Kinetics Rate of chemical

reactions mediated by enzymes Enzymes can increase reaction rate by favoring or

enabling a different reaction pathway with

a lower activation energy making it easier

for the reaction to occur

Michaelis-Menten kinetics

V0 varies with [S]Vmax approachedasymptoticallyV0 is moles of

productformed per sec when [P]is low (close to zero time)E + SESE + P

Michaelis-Menten Model

V0 = Vmax x[S]([S] + Km)

Michaelis-Menten Equation

Determining initial velocity (when [P] is low)

Steady-state amp pre-steady-state conditions

At equilibrium no net change of [S] amp [P]or of [ES] amp [E]

At pre-steady-state[P] is low (close to zero time) hence V0 for initial

reaction velocity

At pre-steady state we can ignore the back reactions

Michaelis-Menten kinetics (summary)

Enzyme kinetics (Michaelis-Menten Graph) At fixed concentration of enzyme V0 is almost linearly proportional to

[S] when [S] is small but is nearly independent of [S] when [S] is large

Proposed Model E + S ES E + P

ES complex is a necessary intermediateObjective find an expression that relates rate of catalysis to theconcentrations of S amp E and the rates of individual steps

Start with V0 = k2[ES] and derive V0 = Vmax x[S]([S] + Km) This equation accounts for graph dataAt low [S] ([S] lt Km) V0 = (VmaxKm)[S]At high [S] ([S] gt Km) V0 = Vmax

When [S] = Km V0 = Vmax2 Thus Km = substrate concentration at which the reaction rate (V0) is half max

Range of Km values

Km provides approximation of [S] in vivo for many enzymes

Lineweaver-Burk plot (double-reciprocal)

Eadie-Hofstee plot

Hanes-Woolf Plot

Allosteric enzymes

Allosteric enzymes tend to be

multi-sub unit proteins

The reversible binding of an

allosteric modulator (here a

positive modulator M) affects

the substrate binding site

T

T

R

T

[S]

vo

Mechanism and Example of Allosteric Effect

S S

R

R

SS

RS

A

I

T[S]

vo

[S]

vo

(+)

(-) X X

X

R = Relax(active)

T = Tense(inactive)

Allosteric siteHomotropic(+)Concerted

Heterotropic(+)Sequential

Heterotropic(-)Concerted

Allosteric site

Kinetics Cooperation Models

(-)

(+)

(+)

Enzyme Inhibitors

bull Specific enzyme inhibitors regulate enzyme activity and help us

understand mechanism of enzyme action (Denaturing agents are not

inhibitors)

bull Irreversible inhibitors form covalent or very tight permanent bonds with

aa at the active site of the enzyme and render it inactive 3 classes

groupspecific reagents substrate analogs suicide inhibitors

bull Reversible inhibitors form an EI complex that can be dissociated back

to enzyme and free inhibitor 3 groups based on their mechanism of

action competitive non-competitive and uncompetitive

Enzyme Inhibition

Competitive inhibitors

bull Compete with substrate for binding to enzyme

bull E + S = ES or E + I = EI Both S and I cannot bind enzyme at the same time

bull In presence of I the equilibrium of E + S = ES is shifted to the left causing dissociation of ES

bull This can be reversed corrected by increasing [S]

bull Vmax is not changed KM is increased by (1 + IKi)

bull Eg AZT antibacterial sulfonamides the anticancer agent methotrexate etc

Competitive Inhibition

Kinetics of competitive inhibitor

Increase [S] toovercomeinhibition

Vmax attainable

Km is increased

Ki =dissociationconstant forinhibitor

V max unaltered Km increased

Non-competitive Inhibitors

bull Inhibitor binding site is distinct from substrate binding site Can bind to free enzyme E and to ES

bull E + I = EI ES + I = ESI or EI + S = ESI

bull Both EI and ESI are enzymatically inactive

bull The effective functional [E] (and [S]) is reduced

bull Reaction of unaffected ES proceeds normally

bull Inhibition cannot be reversed by increasing [S]

bull KM is not changed Vmax is decreased by (1 + IKi)

Mixed (Noncompetitive) Inhibition

Kinetics of non-competitive inhibitor

Increasing [S] cannotovercome inhibition

Less E availableV max is lowerKm remains the samefor available E

Km unaltered V max decreased

Uncompetitive Inhibitors

bull The inhibitor cannot bind to the enzyme directly but can only bind to the enzyme-substrate complex

bull ES + I = ESI

bull Both Vmax and KM are decreased by (1+IKi)

Uncompetitive Inhibition

Substrate Inhibition

Caused by high substrate concentrations

E + S ES E + PKm

rsquo k2

KS1

+

S

ES21

2

2

][][

][

][

]][[

][

]][[

Sm

m

mSi

KS

SK

SVv

ES

ESK

ES

ESSK

Substrate Inhibition

At low substrate concentrations [S]2Ks1ltlt1 and inhibition is not observed

Plot of 1v vs 1[S] gives a line Slope = Krsquo

mVm

Intercept = 1Vm

][

111

][1

SV

K

Vv

SK

Vv

m

m

m

m

m

Substrate Inhibition

At high substrate concentrations Krsquom[S]ltlt1 and

inhibition is dominant

Plot of 1v vs [S] gives a straight line Slope = 1KS1 middot Vm

Intercept = 1Vm

mSm

S

m

VK

S

Vv

KS

Vv

1

1

][11

][1

1

max][

0][

SmKKS

Sddv

1V Igt0

I=0

1Vm

-1Km -1Kmapp 1[S]

1VIgt0

I=0

1Vm

-1Km-1Kmapp 1[S]

1Vmapp

1VIgt0

I=0

1Vm

-1Km 1[S]

1Vmapp

1V

1Vm

-1Km 1[S]

Competitive Uncompetitive

Non-Competitive Substrate Inhibition

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] onlyand competes with [S]increasing [S] overcomesInhibition by [II]

[II] binds to free [E] or [ES] complex Increasing [S] cannot overcome [II] inhibition

[II] binds to [ES] complex only increasing [S] favorsthe inhibition by [II]

E + S rarr ES rarr E + P + IIdarrEII

larr

uarr

E + S rarr ES rarr E + P + + II IIdarr darrEII + S rarrEIIS

larr

uarr uarr

E + S rarr ES rarr E + P + II darr EIIS

larr

uarr

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Kmrsquo [S] mM

vo

[S] mM

vo

II II

Km [S] mM

Vmax

II

Kmrsquo

VmaxrsquoVmaxrsquo

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax amp Km decreased

II

1[S]1Km

1vo

1 Vmax

II

Two parallellines

II

Intersect at X axis

1vo

1 Vmax

1[S]1Km 1[S]1Km

1 Vmax

1vo

Intersect at Y axis

= Kmrsquo

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

Lock and Key Model

An enzyme binds a substrate in a region called the active site

Only certain substrates can fit the active site

Amino acid R groups in the active site help substrate bind

Enzyme-substrate complex forms

Substrate reacts to form product

Product is released

Enzyme Kinetics

- Kinetics The study of the rate of change

- Enzyme Kinetics Rate of chemical

reactions mediated by enzymes Enzymes can increase reaction rate by favoring or

enabling a different reaction pathway with

a lower activation energy making it easier

for the reaction to occur

Michaelis-Menten kinetics

V0 varies with [S]Vmax approachedasymptoticallyV0 is moles of

productformed per sec when [P]is low (close to zero time)E + SESE + P

Michaelis-Menten Model

V0 = Vmax x[S]([S] + Km)

Michaelis-Menten Equation

Determining initial velocity (when [P] is low)

Steady-state amp pre-steady-state conditions

At equilibrium no net change of [S] amp [P]or of [ES] amp [E]

At pre-steady-state[P] is low (close to zero time) hence V0 for initial

reaction velocity

At pre-steady state we can ignore the back reactions

Michaelis-Menten kinetics (summary)

Enzyme kinetics (Michaelis-Menten Graph) At fixed concentration of enzyme V0 is almost linearly proportional to

[S] when [S] is small but is nearly independent of [S] when [S] is large

Proposed Model E + S ES E + P

ES complex is a necessary intermediateObjective find an expression that relates rate of catalysis to theconcentrations of S amp E and the rates of individual steps

Start with V0 = k2[ES] and derive V0 = Vmax x[S]([S] + Km) This equation accounts for graph dataAt low [S] ([S] lt Km) V0 = (VmaxKm)[S]At high [S] ([S] gt Km) V0 = Vmax

When [S] = Km V0 = Vmax2 Thus Km = substrate concentration at which the reaction rate (V0) is half max

Range of Km values

Km provides approximation of [S] in vivo for many enzymes

Lineweaver-Burk plot (double-reciprocal)

Eadie-Hofstee plot

Hanes-Woolf Plot

Allosteric enzymes

Allosteric enzymes tend to be

multi-sub unit proteins

The reversible binding of an

allosteric modulator (here a

positive modulator M) affects

the substrate binding site

T

T

R

T

[S]

vo

Mechanism and Example of Allosteric Effect

S S

R

R

SS

RS

A

I

T[S]

vo

[S]

vo

(+)

(-) X X

X

R = Relax(active)

T = Tense(inactive)

Allosteric siteHomotropic(+)Concerted

Heterotropic(+)Sequential

Heterotropic(-)Concerted

Allosteric site

Kinetics Cooperation Models

(-)

(+)

(+)

Enzyme Inhibitors

bull Specific enzyme inhibitors regulate enzyme activity and help us

understand mechanism of enzyme action (Denaturing agents are not

inhibitors)

bull Irreversible inhibitors form covalent or very tight permanent bonds with

aa at the active site of the enzyme and render it inactive 3 classes

groupspecific reagents substrate analogs suicide inhibitors

bull Reversible inhibitors form an EI complex that can be dissociated back

to enzyme and free inhibitor 3 groups based on their mechanism of

action competitive non-competitive and uncompetitive

Enzyme Inhibition

Competitive inhibitors

bull Compete with substrate for binding to enzyme

bull E + S = ES or E + I = EI Both S and I cannot bind enzyme at the same time

bull In presence of I the equilibrium of E + S = ES is shifted to the left causing dissociation of ES

bull This can be reversed corrected by increasing [S]

bull Vmax is not changed KM is increased by (1 + IKi)

bull Eg AZT antibacterial sulfonamides the anticancer agent methotrexate etc

Competitive Inhibition

Kinetics of competitive inhibitor

Increase [S] toovercomeinhibition

Vmax attainable

Km is increased

Ki =dissociationconstant forinhibitor

V max unaltered Km increased

Non-competitive Inhibitors

bull Inhibitor binding site is distinct from substrate binding site Can bind to free enzyme E and to ES

bull E + I = EI ES + I = ESI or EI + S = ESI

bull Both EI and ESI are enzymatically inactive

bull The effective functional [E] (and [S]) is reduced

bull Reaction of unaffected ES proceeds normally

bull Inhibition cannot be reversed by increasing [S]

bull KM is not changed Vmax is decreased by (1 + IKi)

Mixed (Noncompetitive) Inhibition

Kinetics of non-competitive inhibitor

Increasing [S] cannotovercome inhibition

Less E availableV max is lowerKm remains the samefor available E

Km unaltered V max decreased

Uncompetitive Inhibitors

bull The inhibitor cannot bind to the enzyme directly but can only bind to the enzyme-substrate complex

bull ES + I = ESI

bull Both Vmax and KM are decreased by (1+IKi)

Uncompetitive Inhibition

Substrate Inhibition

Caused by high substrate concentrations

E + S ES E + PKm

rsquo k2

KS1

+

S

ES21

2

2

][][

][

][

]][[

][

]][[

Sm

m

mSi

KS

SK

SVv

ES

ESK

ES

ESSK

Substrate Inhibition

At low substrate concentrations [S]2Ks1ltlt1 and inhibition is not observed

Plot of 1v vs 1[S] gives a line Slope = Krsquo

mVm

Intercept = 1Vm

][

111

][1

SV

K

Vv

SK

Vv

m

m

m

m

m

Substrate Inhibition

At high substrate concentrations Krsquom[S]ltlt1 and

inhibition is dominant

Plot of 1v vs [S] gives a straight line Slope = 1KS1 middot Vm

Intercept = 1Vm

mSm

S

m

VK

S

Vv

KS

Vv

1

1

][11

][1

1

max][

0][

SmKKS

Sddv

1V Igt0

I=0

1Vm

-1Km -1Kmapp 1[S]

1VIgt0

I=0

1Vm

-1Km-1Kmapp 1[S]

1Vmapp

1VIgt0

I=0

1Vm

-1Km 1[S]

1Vmapp

1V

1Vm

-1Km 1[S]

Competitive Uncompetitive

Non-Competitive Substrate Inhibition

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] onlyand competes with [S]increasing [S] overcomesInhibition by [II]

[II] binds to free [E] or [ES] complex Increasing [S] cannot overcome [II] inhibition

[II] binds to [ES] complex only increasing [S] favorsthe inhibition by [II]

E + S rarr ES rarr E + P + IIdarrEII

larr

uarr

E + S rarr ES rarr E + P + + II IIdarr darrEII + S rarrEIIS

larr

uarr uarr

E + S rarr ES rarr E + P + II darr EIIS

larr

uarr

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Kmrsquo [S] mM

vo

[S] mM

vo

II II

Km [S] mM

Vmax

II

Kmrsquo

VmaxrsquoVmaxrsquo

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax amp Km decreased

II

1[S]1Km

1vo

1 Vmax

II

Two parallellines

II

Intersect at X axis

1vo

1 Vmax

1[S]1Km 1[S]1Km

1 Vmax

1vo

Intersect at Y axis

= Kmrsquo

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

Enzyme Kinetics

- Kinetics The study of the rate of change

- Enzyme Kinetics Rate of chemical

reactions mediated by enzymes Enzymes can increase reaction rate by favoring or

enabling a different reaction pathway with

a lower activation energy making it easier

for the reaction to occur

Michaelis-Menten kinetics

V0 varies with [S]Vmax approachedasymptoticallyV0 is moles of

productformed per sec when [P]is low (close to zero time)E + SESE + P

Michaelis-Menten Model

V0 = Vmax x[S]([S] + Km)

Michaelis-Menten Equation

Determining initial velocity (when [P] is low)

Steady-state amp pre-steady-state conditions

At equilibrium no net change of [S] amp [P]or of [ES] amp [E]

At pre-steady-state[P] is low (close to zero time) hence V0 for initial

reaction velocity

At pre-steady state we can ignore the back reactions

Michaelis-Menten kinetics (summary)

Enzyme kinetics (Michaelis-Menten Graph) At fixed concentration of enzyme V0 is almost linearly proportional to

[S] when [S] is small but is nearly independent of [S] when [S] is large

Proposed Model E + S ES E + P

ES complex is a necessary intermediateObjective find an expression that relates rate of catalysis to theconcentrations of S amp E and the rates of individual steps

Start with V0 = k2[ES] and derive V0 = Vmax x[S]([S] + Km) This equation accounts for graph dataAt low [S] ([S] lt Km) V0 = (VmaxKm)[S]At high [S] ([S] gt Km) V0 = Vmax

When [S] = Km V0 = Vmax2 Thus Km = substrate concentration at which the reaction rate (V0) is half max

Range of Km values

Km provides approximation of [S] in vivo for many enzymes

Lineweaver-Burk plot (double-reciprocal)

Eadie-Hofstee plot

Hanes-Woolf Plot

Allosteric enzymes

Allosteric enzymes tend to be

multi-sub unit proteins

The reversible binding of an

allosteric modulator (here a

positive modulator M) affects

the substrate binding site

T

T

R

T

[S]

vo

Mechanism and Example of Allosteric Effect

S S

R

R

SS

RS

A

I

T[S]

vo

[S]

vo

(+)

(-) X X

X

R = Relax(active)

T = Tense(inactive)

Allosteric siteHomotropic(+)Concerted

Heterotropic(+)Sequential

Heterotropic(-)Concerted

Allosteric site

Kinetics Cooperation Models

(-)

(+)

(+)

Enzyme Inhibitors

bull Specific enzyme inhibitors regulate enzyme activity and help us

understand mechanism of enzyme action (Denaturing agents are not

inhibitors)

bull Irreversible inhibitors form covalent or very tight permanent bonds with

aa at the active site of the enzyme and render it inactive 3 classes

groupspecific reagents substrate analogs suicide inhibitors

bull Reversible inhibitors form an EI complex that can be dissociated back

to enzyme and free inhibitor 3 groups based on their mechanism of

action competitive non-competitive and uncompetitive

Enzyme Inhibition

Competitive inhibitors

bull Compete with substrate for binding to enzyme

bull E + S = ES or E + I = EI Both S and I cannot bind enzyme at the same time

bull In presence of I the equilibrium of E + S = ES is shifted to the left causing dissociation of ES

bull This can be reversed corrected by increasing [S]

bull Vmax is not changed KM is increased by (1 + IKi)

bull Eg AZT antibacterial sulfonamides the anticancer agent methotrexate etc

Competitive Inhibition

Kinetics of competitive inhibitor

Increase [S] toovercomeinhibition

Vmax attainable

Km is increased

Ki =dissociationconstant forinhibitor

V max unaltered Km increased

Non-competitive Inhibitors

bull Inhibitor binding site is distinct from substrate binding site Can bind to free enzyme E and to ES

bull E + I = EI ES + I = ESI or EI + S = ESI

bull Both EI and ESI are enzymatically inactive

bull The effective functional [E] (and [S]) is reduced

bull Reaction of unaffected ES proceeds normally

bull Inhibition cannot be reversed by increasing [S]

bull KM is not changed Vmax is decreased by (1 + IKi)

Mixed (Noncompetitive) Inhibition

Kinetics of non-competitive inhibitor

Increasing [S] cannotovercome inhibition

Less E availableV max is lowerKm remains the samefor available E

Km unaltered V max decreased

Uncompetitive Inhibitors

bull The inhibitor cannot bind to the enzyme directly but can only bind to the enzyme-substrate complex

bull ES + I = ESI

bull Both Vmax and KM are decreased by (1+IKi)

Uncompetitive Inhibition

Substrate Inhibition

Caused by high substrate concentrations

E + S ES E + PKm

rsquo k2

KS1

+

S

ES21

2

2

][][

][

][

]][[

][

]][[

Sm

m

mSi

KS

SK

SVv

ES

ESK

ES

ESSK

Substrate Inhibition

At low substrate concentrations [S]2Ks1ltlt1 and inhibition is not observed

Plot of 1v vs 1[S] gives a line Slope = Krsquo

mVm

Intercept = 1Vm

][

111

][1

SV

K

Vv

SK

Vv

m

m

m

m

m

Substrate Inhibition

At high substrate concentrations Krsquom[S]ltlt1 and

inhibition is dominant

Plot of 1v vs [S] gives a straight line Slope = 1KS1 middot Vm

Intercept = 1Vm

mSm

S

m

VK

S

Vv

KS

Vv

1

1

][11

][1

1

max][

0][

SmKKS

Sddv

1V Igt0

I=0

1Vm

-1Km -1Kmapp 1[S]

1VIgt0

I=0

1Vm

-1Km-1Kmapp 1[S]

1Vmapp

1VIgt0

I=0

1Vm

-1Km 1[S]

1Vmapp

1V

1Vm

-1Km 1[S]

Competitive Uncompetitive

Non-Competitive Substrate Inhibition

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] onlyand competes with [S]increasing [S] overcomesInhibition by [II]

[II] binds to free [E] or [ES] complex Increasing [S] cannot overcome [II] inhibition

[II] binds to [ES] complex only increasing [S] favorsthe inhibition by [II]

E + S rarr ES rarr E + P + IIdarrEII

larr

uarr

E + S rarr ES rarr E + P + + II IIdarr darrEII + S rarrEIIS

larr

uarr uarr

E + S rarr ES rarr E + P + II darr EIIS

larr

uarr

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Kmrsquo [S] mM

vo

[S] mM

vo

II II

Km [S] mM

Vmax

II

Kmrsquo

VmaxrsquoVmaxrsquo

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax amp Km decreased

II

1[S]1Km

1vo

1 Vmax

II

Two parallellines

II

Intersect at X axis

1vo

1 Vmax

1[S]1Km 1[S]1Km

1 Vmax

1vo

Intersect at Y axis

= Kmrsquo

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

Michaelis-Menten kinetics

V0 varies with [S]Vmax approachedasymptoticallyV0 is moles of

productformed per sec when [P]is low (close to zero time)E + SESE + P

Michaelis-Menten Model

V0 = Vmax x[S]([S] + Km)

Michaelis-Menten Equation

Determining initial velocity (when [P] is low)

Steady-state amp pre-steady-state conditions

At equilibrium no net change of [S] amp [P]or of [ES] amp [E]

At pre-steady-state[P] is low (close to zero time) hence V0 for initial

reaction velocity

At pre-steady state we can ignore the back reactions

Michaelis-Menten kinetics (summary)

Enzyme kinetics (Michaelis-Menten Graph) At fixed concentration of enzyme V0 is almost linearly proportional to

[S] when [S] is small but is nearly independent of [S] when [S] is large

Proposed Model E + S ES E + P

ES complex is a necessary intermediateObjective find an expression that relates rate of catalysis to theconcentrations of S amp E and the rates of individual steps

Start with V0 = k2[ES] and derive V0 = Vmax x[S]([S] + Km) This equation accounts for graph dataAt low [S] ([S] lt Km) V0 = (VmaxKm)[S]At high [S] ([S] gt Km) V0 = Vmax

When [S] = Km V0 = Vmax2 Thus Km = substrate concentration at which the reaction rate (V0) is half max

Range of Km values

Km provides approximation of [S] in vivo for many enzymes

Lineweaver-Burk plot (double-reciprocal)

Eadie-Hofstee plot

Hanes-Woolf Plot

Allosteric enzymes

Allosteric enzymes tend to be

multi-sub unit proteins

The reversible binding of an

allosteric modulator (here a

positive modulator M) affects

the substrate binding site

T

T

R

T

[S]

vo

Mechanism and Example of Allosteric Effect

S S

R

R

SS

RS

A

I

T[S]

vo

[S]

vo

(+)

(-) X X

X

R = Relax(active)

T = Tense(inactive)

Allosteric siteHomotropic(+)Concerted

Heterotropic(+)Sequential

Heterotropic(-)Concerted

Allosteric site

Kinetics Cooperation Models

(-)

(+)

(+)

Enzyme Inhibitors

bull Specific enzyme inhibitors regulate enzyme activity and help us

understand mechanism of enzyme action (Denaturing agents are not

inhibitors)

bull Irreversible inhibitors form covalent or very tight permanent bonds with

aa at the active site of the enzyme and render it inactive 3 classes

groupspecific reagents substrate analogs suicide inhibitors

bull Reversible inhibitors form an EI complex that can be dissociated back

to enzyme and free inhibitor 3 groups based on their mechanism of

action competitive non-competitive and uncompetitive

Enzyme Inhibition

Competitive inhibitors

bull Compete with substrate for binding to enzyme

bull E + S = ES or E + I = EI Both S and I cannot bind enzyme at the same time

bull In presence of I the equilibrium of E + S = ES is shifted to the left causing dissociation of ES

bull This can be reversed corrected by increasing [S]

bull Vmax is not changed KM is increased by (1 + IKi)

bull Eg AZT antibacterial sulfonamides the anticancer agent methotrexate etc

Competitive Inhibition

Kinetics of competitive inhibitor

Increase [S] toovercomeinhibition

Vmax attainable

Km is increased

Ki =dissociationconstant forinhibitor

V max unaltered Km increased

Non-competitive Inhibitors

bull Inhibitor binding site is distinct from substrate binding site Can bind to free enzyme E and to ES

bull E + I = EI ES + I = ESI or EI + S = ESI

bull Both EI and ESI are enzymatically inactive

bull The effective functional [E] (and [S]) is reduced

bull Reaction of unaffected ES proceeds normally

bull Inhibition cannot be reversed by increasing [S]

bull KM is not changed Vmax is decreased by (1 + IKi)

Mixed (Noncompetitive) Inhibition

Kinetics of non-competitive inhibitor

Increasing [S] cannotovercome inhibition

Less E availableV max is lowerKm remains the samefor available E

Km unaltered V max decreased

Uncompetitive Inhibitors

bull The inhibitor cannot bind to the enzyme directly but can only bind to the enzyme-substrate complex

bull ES + I = ESI

bull Both Vmax and KM are decreased by (1+IKi)

Uncompetitive Inhibition

Substrate Inhibition

Caused by high substrate concentrations

E + S ES E + PKm

rsquo k2

KS1

+

S

ES21

2

2

][][

][

][

]][[

][

]][[

Sm

m

mSi

KS

SK

SVv

ES

ESK

ES

ESSK

Substrate Inhibition

At low substrate concentrations [S]2Ks1ltlt1 and inhibition is not observed

Plot of 1v vs 1[S] gives a line Slope = Krsquo

mVm

Intercept = 1Vm

][

111

][1

SV

K

Vv

SK

Vv

m

m

m

m

m

Substrate Inhibition

At high substrate concentrations Krsquom[S]ltlt1 and

inhibition is dominant

Plot of 1v vs [S] gives a straight line Slope = 1KS1 middot Vm

Intercept = 1Vm

mSm

S

m

VK

S

Vv

KS

Vv

1

1

][11

][1

1

max][

0][

SmKKS

Sddv

1V Igt0

I=0

1Vm

-1Km -1Kmapp 1[S]

1VIgt0

I=0

1Vm

-1Km-1Kmapp 1[S]

1Vmapp

1VIgt0

I=0

1Vm

-1Km 1[S]

1Vmapp

1V

1Vm

-1Km 1[S]

Competitive Uncompetitive

Non-Competitive Substrate Inhibition

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] onlyand competes with [S]increasing [S] overcomesInhibition by [II]

[II] binds to free [E] or [ES] complex Increasing [S] cannot overcome [II] inhibition

[II] binds to [ES] complex only increasing [S] favorsthe inhibition by [II]

E + S rarr ES rarr E + P + IIdarrEII

larr

uarr

E + S rarr ES rarr E + P + + II IIdarr darrEII + S rarrEIIS

larr

uarr uarr

E + S rarr ES rarr E + P + II darr EIIS

larr

uarr

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Kmrsquo [S] mM

vo

[S] mM

vo

II II

Km [S] mM

Vmax

II

Kmrsquo

VmaxrsquoVmaxrsquo

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax amp Km decreased

II

1[S]1Km

1vo

1 Vmax

II

Two parallellines

II

Intersect at X axis

1vo

1 Vmax

1[S]1Km 1[S]1Km

1 Vmax

1vo

Intersect at Y axis

= Kmrsquo

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

Determining initial velocity (when [P] is low)

Steady-state amp pre-steady-state conditions

At equilibrium no net change of [S] amp [P]or of [ES] amp [E]

At pre-steady-state[P] is low (close to zero time) hence V0 for initial

reaction velocity

At pre-steady state we can ignore the back reactions

Michaelis-Menten kinetics (summary)

Enzyme kinetics (Michaelis-Menten Graph) At fixed concentration of enzyme V0 is almost linearly proportional to

[S] when [S] is small but is nearly independent of [S] when [S] is large

Proposed Model E + S ES E + P

ES complex is a necessary intermediateObjective find an expression that relates rate of catalysis to theconcentrations of S amp E and the rates of individual steps

Start with V0 = k2[ES] and derive V0 = Vmax x[S]([S] + Km) This equation accounts for graph dataAt low [S] ([S] lt Km) V0 = (VmaxKm)[S]At high [S] ([S] gt Km) V0 = Vmax

When [S] = Km V0 = Vmax2 Thus Km = substrate concentration at which the reaction rate (V0) is half max

Range of Km values

Km provides approximation of [S] in vivo for many enzymes

Lineweaver-Burk plot (double-reciprocal)

Eadie-Hofstee plot

Hanes-Woolf Plot

Allosteric enzymes

Allosteric enzymes tend to be

multi-sub unit proteins

The reversible binding of an

allosteric modulator (here a

positive modulator M) affects

the substrate binding site

T

T

R

T

[S]

vo

Mechanism and Example of Allosteric Effect

S S

R

R

SS

RS

A

I

T[S]

vo

[S]

vo

(+)

(-) X X

X

R = Relax(active)

T = Tense(inactive)

Allosteric siteHomotropic(+)Concerted

Heterotropic(+)Sequential

Heterotropic(-)Concerted

Allosteric site

Kinetics Cooperation Models

(-)

(+)

(+)

Enzyme Inhibitors

bull Specific enzyme inhibitors regulate enzyme activity and help us

understand mechanism of enzyme action (Denaturing agents are not

inhibitors)

bull Irreversible inhibitors form covalent or very tight permanent bonds with

aa at the active site of the enzyme and render it inactive 3 classes

groupspecific reagents substrate analogs suicide inhibitors

bull Reversible inhibitors form an EI complex that can be dissociated back

to enzyme and free inhibitor 3 groups based on their mechanism of

action competitive non-competitive and uncompetitive

Enzyme Inhibition

Competitive inhibitors

bull Compete with substrate for binding to enzyme

bull E + S = ES or E + I = EI Both S and I cannot bind enzyme at the same time

bull In presence of I the equilibrium of E + S = ES is shifted to the left causing dissociation of ES

bull This can be reversed corrected by increasing [S]

bull Vmax is not changed KM is increased by (1 + IKi)

bull Eg AZT antibacterial sulfonamides the anticancer agent methotrexate etc

Competitive Inhibition

Kinetics of competitive inhibitor

Increase [S] toovercomeinhibition

Vmax attainable

Km is increased

Ki =dissociationconstant forinhibitor

V max unaltered Km increased

Non-competitive Inhibitors

bull Inhibitor binding site is distinct from substrate binding site Can bind to free enzyme E and to ES

bull E + I = EI ES + I = ESI or EI + S = ESI

bull Both EI and ESI are enzymatically inactive

bull The effective functional [E] (and [S]) is reduced

bull Reaction of unaffected ES proceeds normally

bull Inhibition cannot be reversed by increasing [S]

bull KM is not changed Vmax is decreased by (1 + IKi)

Mixed (Noncompetitive) Inhibition

Kinetics of non-competitive inhibitor

Increasing [S] cannotovercome inhibition

Less E availableV max is lowerKm remains the samefor available E

Km unaltered V max decreased

Uncompetitive Inhibitors

bull The inhibitor cannot bind to the enzyme directly but can only bind to the enzyme-substrate complex

bull ES + I = ESI

bull Both Vmax and KM are decreased by (1+IKi)

Uncompetitive Inhibition

Substrate Inhibition

Caused by high substrate concentrations

E + S ES E + PKm

rsquo k2

KS1

+

S

ES21

2

2

][][

][

][

]][[

][

]][[

Sm

m

mSi

KS

SK

SVv

ES

ESK

ES

ESSK

Substrate Inhibition

At low substrate concentrations [S]2Ks1ltlt1 and inhibition is not observed

Plot of 1v vs 1[S] gives a line Slope = Krsquo

mVm

Intercept = 1Vm

][

111

][1

SV

K

Vv

SK

Vv

m

m

m

m

m

Substrate Inhibition

At high substrate concentrations Krsquom[S]ltlt1 and

inhibition is dominant

Plot of 1v vs [S] gives a straight line Slope = 1KS1 middot Vm

Intercept = 1Vm

mSm

S

m

VK

S

Vv

KS

Vv

1

1

][11

][1

1

max][

0][

SmKKS

Sddv

1V Igt0

I=0

1Vm

-1Km -1Kmapp 1[S]

1VIgt0

I=0

1Vm

-1Km-1Kmapp 1[S]

1Vmapp

1VIgt0

I=0

1Vm

-1Km 1[S]

1Vmapp

1V

1Vm

-1Km 1[S]

Competitive Uncompetitive

Non-Competitive Substrate Inhibition

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] onlyand competes with [S]increasing [S] overcomesInhibition by [II]

[II] binds to free [E] or [ES] complex Increasing [S] cannot overcome [II] inhibition

[II] binds to [ES] complex only increasing [S] favorsthe inhibition by [II]

E + S rarr ES rarr E + P + IIdarrEII

larr

uarr

E + S rarr ES rarr E + P + + II IIdarr darrEII + S rarrEIIS

larr

uarr uarr

E + S rarr ES rarr E + P + II darr EIIS

larr

uarr

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Kmrsquo [S] mM

vo

[S] mM

vo

II II

Km [S] mM

Vmax

II

Kmrsquo

VmaxrsquoVmaxrsquo

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax amp Km decreased

II

1[S]1Km

1vo

1 Vmax

II

Two parallellines

II

Intersect at X axis

1vo

1 Vmax

1[S]1Km 1[S]1Km

1 Vmax

1vo

Intersect at Y axis

= Kmrsquo

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

Steady-state amp pre-steady-state conditions

At equilibrium no net change of [S] amp [P]or of [ES] amp [E]

At pre-steady-state[P] is low (close to zero time) hence V0 for initial

reaction velocity

At pre-steady state we can ignore the back reactions

Michaelis-Menten kinetics (summary)

Enzyme kinetics (Michaelis-Menten Graph) At fixed concentration of enzyme V0 is almost linearly proportional to

[S] when [S] is small but is nearly independent of [S] when [S] is large

Proposed Model E + S ES E + P

ES complex is a necessary intermediateObjective find an expression that relates rate of catalysis to theconcentrations of S amp E and the rates of individual steps

Start with V0 = k2[ES] and derive V0 = Vmax x[S]([S] + Km) This equation accounts for graph dataAt low [S] ([S] lt Km) V0 = (VmaxKm)[S]At high [S] ([S] gt Km) V0 = Vmax

When [S] = Km V0 = Vmax2 Thus Km = substrate concentration at which the reaction rate (V0) is half max

Range of Km values

Km provides approximation of [S] in vivo for many enzymes

Lineweaver-Burk plot (double-reciprocal)

Eadie-Hofstee plot

Hanes-Woolf Plot

Allosteric enzymes

Allosteric enzymes tend to be

multi-sub unit proteins

The reversible binding of an

allosteric modulator (here a

positive modulator M) affects

the substrate binding site

T

T

R

T

[S]

vo

Mechanism and Example of Allosteric Effect

S S

R

R

SS

RS

A

I

T[S]

vo

[S]

vo

(+)

(-) X X

X

R = Relax(active)

T = Tense(inactive)

Allosteric siteHomotropic(+)Concerted

Heterotropic(+)Sequential

Heterotropic(-)Concerted

Allosteric site

Kinetics Cooperation Models

(-)

(+)

(+)

Enzyme Inhibitors

bull Specific enzyme inhibitors regulate enzyme activity and help us

understand mechanism of enzyme action (Denaturing agents are not

inhibitors)

bull Irreversible inhibitors form covalent or very tight permanent bonds with

aa at the active site of the enzyme and render it inactive 3 classes

groupspecific reagents substrate analogs suicide inhibitors

bull Reversible inhibitors form an EI complex that can be dissociated back

to enzyme and free inhibitor 3 groups based on their mechanism of

action competitive non-competitive and uncompetitive

Enzyme Inhibition

Competitive inhibitors

bull Compete with substrate for binding to enzyme

bull E + S = ES or E + I = EI Both S and I cannot bind enzyme at the same time

bull In presence of I the equilibrium of E + S = ES is shifted to the left causing dissociation of ES

bull This can be reversed corrected by increasing [S]

bull Vmax is not changed KM is increased by (1 + IKi)

bull Eg AZT antibacterial sulfonamides the anticancer agent methotrexate etc

Competitive Inhibition

Kinetics of competitive inhibitor

Increase [S] toovercomeinhibition

Vmax attainable

Km is increased

Ki =dissociationconstant forinhibitor

V max unaltered Km increased

Non-competitive Inhibitors

bull Inhibitor binding site is distinct from substrate binding site Can bind to free enzyme E and to ES

bull E + I = EI ES + I = ESI or EI + S = ESI

bull Both EI and ESI are enzymatically inactive

bull The effective functional [E] (and [S]) is reduced

bull Reaction of unaffected ES proceeds normally

bull Inhibition cannot be reversed by increasing [S]

bull KM is not changed Vmax is decreased by (1 + IKi)

Mixed (Noncompetitive) Inhibition

Kinetics of non-competitive inhibitor

Increasing [S] cannotovercome inhibition

Less E availableV max is lowerKm remains the samefor available E

Km unaltered V max decreased

Uncompetitive Inhibitors

bull The inhibitor cannot bind to the enzyme directly but can only bind to the enzyme-substrate complex

bull ES + I = ESI

bull Both Vmax and KM are decreased by (1+IKi)

Uncompetitive Inhibition

Substrate Inhibition

Caused by high substrate concentrations

E + S ES E + PKm

rsquo k2

KS1

+

S

ES21

2

2

][][

][

][

]][[

][

]][[

Sm

m

mSi

KS

SK

SVv

ES

ESK

ES

ESSK

Substrate Inhibition

At low substrate concentrations [S]2Ks1ltlt1 and inhibition is not observed

Plot of 1v vs 1[S] gives a line Slope = Krsquo

mVm

Intercept = 1Vm

][

111

][1

SV

K

Vv

SK

Vv

m

m

m

m

m

Substrate Inhibition

At high substrate concentrations Krsquom[S]ltlt1 and

inhibition is dominant

Plot of 1v vs [S] gives a straight line Slope = 1KS1 middot Vm

Intercept = 1Vm

mSm

S

m

VK

S

Vv

KS

Vv

1

1

][11

][1

1

max][

0][

SmKKS

Sddv

1V Igt0

I=0

1Vm

-1Km -1Kmapp 1[S]

1VIgt0

I=0

1Vm

-1Km-1Kmapp 1[S]

1Vmapp

1VIgt0

I=0

1Vm

-1Km 1[S]

1Vmapp

1V

1Vm

-1Km 1[S]

Competitive Uncompetitive

Non-Competitive Substrate Inhibition

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] onlyand competes with [S]increasing [S] overcomesInhibition by [II]

[II] binds to free [E] or [ES] complex Increasing [S] cannot overcome [II] inhibition

[II] binds to [ES] complex only increasing [S] favorsthe inhibition by [II]

E + S rarr ES rarr E + P + IIdarrEII

larr

uarr

E + S rarr ES rarr E + P + + II IIdarr darrEII + S rarrEIIS

larr

uarr uarr

E + S rarr ES rarr E + P + II darr EIIS

larr

uarr

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Kmrsquo [S] mM

vo

[S] mM

vo

II II

Km [S] mM

Vmax

II

Kmrsquo

VmaxrsquoVmaxrsquo

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax amp Km decreased

II

1[S]1Km

1vo

1 Vmax

II

Two parallellines

II

Intersect at X axis

1vo

1 Vmax

1[S]1Km 1[S]1Km

1 Vmax

1vo

Intersect at Y axis

= Kmrsquo

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

Michaelis-Menten kinetics (summary)

Enzyme kinetics (Michaelis-Menten Graph) At fixed concentration of enzyme V0 is almost linearly proportional to

[S] when [S] is small but is nearly independent of [S] when [S] is large

Proposed Model E + S ES E + P

ES complex is a necessary intermediateObjective find an expression that relates rate of catalysis to theconcentrations of S amp E and the rates of individual steps

Start with V0 = k2[ES] and derive V0 = Vmax x[S]([S] + Km) This equation accounts for graph dataAt low [S] ([S] lt Km) V0 = (VmaxKm)[S]At high [S] ([S] gt Km) V0 = Vmax

When [S] = Km V0 = Vmax2 Thus Km = substrate concentration at which the reaction rate (V0) is half max

Range of Km values

Km provides approximation of [S] in vivo for many enzymes

Lineweaver-Burk plot (double-reciprocal)

Eadie-Hofstee plot

Hanes-Woolf Plot

Allosteric enzymes

Allosteric enzymes tend to be

multi-sub unit proteins

The reversible binding of an

allosteric modulator (here a

positive modulator M) affects

the substrate binding site

T

T

R

T

[S]

vo

Mechanism and Example of Allosteric Effect

S S

R

R

SS

RS

A

I

T[S]

vo

[S]

vo

(+)

(-) X X

X

R = Relax(active)

T = Tense(inactive)

Allosteric siteHomotropic(+)Concerted

Heterotropic(+)Sequential

Heterotropic(-)Concerted

Allosteric site

Kinetics Cooperation Models

(-)

(+)

(+)

Enzyme Inhibitors

bull Specific enzyme inhibitors regulate enzyme activity and help us

understand mechanism of enzyme action (Denaturing agents are not

inhibitors)

bull Irreversible inhibitors form covalent or very tight permanent bonds with

aa at the active site of the enzyme and render it inactive 3 classes

groupspecific reagents substrate analogs suicide inhibitors

bull Reversible inhibitors form an EI complex that can be dissociated back

to enzyme and free inhibitor 3 groups based on their mechanism of

action competitive non-competitive and uncompetitive

Enzyme Inhibition

Competitive inhibitors

bull Compete with substrate for binding to enzyme

bull E + S = ES or E + I = EI Both S and I cannot bind enzyme at the same time

bull In presence of I the equilibrium of E + S = ES is shifted to the left causing dissociation of ES

bull This can be reversed corrected by increasing [S]

bull Vmax is not changed KM is increased by (1 + IKi)

bull Eg AZT antibacterial sulfonamides the anticancer agent methotrexate etc

Competitive Inhibition

Kinetics of competitive inhibitor

Increase [S] toovercomeinhibition

Vmax attainable

Km is increased

Ki =dissociationconstant forinhibitor

V max unaltered Km increased

Non-competitive Inhibitors

bull Inhibitor binding site is distinct from substrate binding site Can bind to free enzyme E and to ES

bull E + I = EI ES + I = ESI or EI + S = ESI

bull Both EI and ESI are enzymatically inactive

bull The effective functional [E] (and [S]) is reduced

bull Reaction of unaffected ES proceeds normally

bull Inhibition cannot be reversed by increasing [S]

bull KM is not changed Vmax is decreased by (1 + IKi)

Mixed (Noncompetitive) Inhibition

Kinetics of non-competitive inhibitor

Increasing [S] cannotovercome inhibition

Less E availableV max is lowerKm remains the samefor available E

Km unaltered V max decreased

Uncompetitive Inhibitors

bull The inhibitor cannot bind to the enzyme directly but can only bind to the enzyme-substrate complex

bull ES + I = ESI

bull Both Vmax and KM are decreased by (1+IKi)

Uncompetitive Inhibition

Substrate Inhibition

Caused by high substrate concentrations

E + S ES E + PKm

rsquo k2

KS1

+

S

ES21

2

2

][][

][

][

]][[

][

]][[

Sm

m

mSi

KS

SK

SVv

ES

ESK

ES

ESSK

Substrate Inhibition

At low substrate concentrations [S]2Ks1ltlt1 and inhibition is not observed

Plot of 1v vs 1[S] gives a line Slope = Krsquo

mVm

Intercept = 1Vm

][

111

][1

SV

K

Vv

SK

Vv

m

m

m

m

m

Substrate Inhibition

At high substrate concentrations Krsquom[S]ltlt1 and

inhibition is dominant

Plot of 1v vs [S] gives a straight line Slope = 1KS1 middot Vm

Intercept = 1Vm

mSm

S

m

VK

S

Vv

KS

Vv

1

1

][11

][1

1

max][

0][

SmKKS

Sddv

1V Igt0

I=0

1Vm

-1Km -1Kmapp 1[S]

1VIgt0

I=0

1Vm

-1Km-1Kmapp 1[S]

1Vmapp

1VIgt0

I=0

1Vm

-1Km 1[S]

1Vmapp

1V

1Vm

-1Km 1[S]

Competitive Uncompetitive

Non-Competitive Substrate Inhibition

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] onlyand competes with [S]increasing [S] overcomesInhibition by [II]

[II] binds to free [E] or [ES] complex Increasing [S] cannot overcome [II] inhibition

[II] binds to [ES] complex only increasing [S] favorsthe inhibition by [II]

E + S rarr ES rarr E + P + IIdarrEII

larr

uarr

E + S rarr ES rarr E + P + + II IIdarr darrEII + S rarrEIIS

larr

uarr uarr

E + S rarr ES rarr E + P + II darr EIIS

larr

uarr

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Kmrsquo [S] mM

vo

[S] mM

vo

II II

Km [S] mM

Vmax

II

Kmrsquo

VmaxrsquoVmaxrsquo

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax amp Km decreased

II

1[S]1Km

1vo

1 Vmax

II

Two parallellines

II

Intersect at X axis

1vo

1 Vmax

1[S]1Km 1[S]1Km

1 Vmax

1vo

Intersect at Y axis

= Kmrsquo

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

Range of Km values

Km provides approximation of [S] in vivo for many enzymes

Lineweaver-Burk plot (double-reciprocal)

Eadie-Hofstee plot

Hanes-Woolf Plot

Allosteric enzymes

Allosteric enzymes tend to be

multi-sub unit proteins

The reversible binding of an

allosteric modulator (here a

positive modulator M) affects

the substrate binding site

T

T

R

T

[S]

vo

Mechanism and Example of Allosteric Effect

S S

R

R

SS

RS

A

I

T[S]

vo

[S]

vo

(+)

(-) X X

X

R = Relax(active)

T = Tense(inactive)

Allosteric siteHomotropic(+)Concerted

Heterotropic(+)Sequential

Heterotropic(-)Concerted

Allosteric site

Kinetics Cooperation Models

(-)

(+)

(+)

Enzyme Inhibitors

bull Specific enzyme inhibitors regulate enzyme activity and help us

understand mechanism of enzyme action (Denaturing agents are not

inhibitors)

bull Irreversible inhibitors form covalent or very tight permanent bonds with

aa at the active site of the enzyme and render it inactive 3 classes

groupspecific reagents substrate analogs suicide inhibitors

bull Reversible inhibitors form an EI complex that can be dissociated back

to enzyme and free inhibitor 3 groups based on their mechanism of

action competitive non-competitive and uncompetitive

Enzyme Inhibition

Competitive inhibitors

bull Compete with substrate for binding to enzyme

bull E + S = ES or E + I = EI Both S and I cannot bind enzyme at the same time

bull In presence of I the equilibrium of E + S = ES is shifted to the left causing dissociation of ES

bull This can be reversed corrected by increasing [S]

bull Vmax is not changed KM is increased by (1 + IKi)

bull Eg AZT antibacterial sulfonamides the anticancer agent methotrexate etc

Competitive Inhibition

Kinetics of competitive inhibitor

Increase [S] toovercomeinhibition

Vmax attainable

Km is increased

Ki =dissociationconstant forinhibitor

V max unaltered Km increased

Non-competitive Inhibitors

bull Inhibitor binding site is distinct from substrate binding site Can bind to free enzyme E and to ES

bull E + I = EI ES + I = ESI or EI + S = ESI

bull Both EI and ESI are enzymatically inactive

bull The effective functional [E] (and [S]) is reduced

bull Reaction of unaffected ES proceeds normally

bull Inhibition cannot be reversed by increasing [S]

bull KM is not changed Vmax is decreased by (1 + IKi)

Mixed (Noncompetitive) Inhibition

Kinetics of non-competitive inhibitor

Increasing [S] cannotovercome inhibition

Less E availableV max is lowerKm remains the samefor available E

Km unaltered V max decreased

Uncompetitive Inhibitors

bull The inhibitor cannot bind to the enzyme directly but can only bind to the enzyme-substrate complex

bull ES + I = ESI

bull Both Vmax and KM are decreased by (1+IKi)

Uncompetitive Inhibition

Substrate Inhibition

Caused by high substrate concentrations

E + S ES E + PKm

rsquo k2

KS1

+

S

ES21

2

2

][][

][

][

]][[

][

]][[

Sm

m

mSi

KS

SK

SVv

ES

ESK

ES

ESSK

Substrate Inhibition

At low substrate concentrations [S]2Ks1ltlt1 and inhibition is not observed

Plot of 1v vs 1[S] gives a line Slope = Krsquo

mVm

Intercept = 1Vm

][

111

][1

SV

K

Vv

SK

Vv

m

m

m

m

m

Substrate Inhibition

At high substrate concentrations Krsquom[S]ltlt1 and

inhibition is dominant

Plot of 1v vs [S] gives a straight line Slope = 1KS1 middot Vm

Intercept = 1Vm

mSm

S

m

VK

S

Vv

KS

Vv

1

1

][11

][1

1

max][

0][

SmKKS

Sddv

1V Igt0

I=0

1Vm

-1Km -1Kmapp 1[S]

1VIgt0

I=0

1Vm

-1Km-1Kmapp 1[S]

1Vmapp

1VIgt0

I=0

1Vm

-1Km 1[S]

1Vmapp

1V

1Vm

-1Km 1[S]

Competitive Uncompetitive

Non-Competitive Substrate Inhibition

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] onlyand competes with [S]increasing [S] overcomesInhibition by [II]

[II] binds to free [E] or [ES] complex Increasing [S] cannot overcome [II] inhibition

[II] binds to [ES] complex only increasing [S] favorsthe inhibition by [II]

E + S rarr ES rarr E + P + IIdarrEII

larr

uarr

E + S rarr ES rarr E + P + + II IIdarr darrEII + S rarrEIIS

larr

uarr uarr

E + S rarr ES rarr E + P + II darr EIIS

larr

uarr

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Kmrsquo [S] mM

vo

[S] mM

vo

II II

Km [S] mM

Vmax

II

Kmrsquo

VmaxrsquoVmaxrsquo

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax amp Km decreased

II

1[S]1Km

1vo

1 Vmax

II

Two parallellines

II

Intersect at X axis

1vo

1 Vmax

1[S]1Km 1[S]1Km

1 Vmax

1vo

Intersect at Y axis

= Kmrsquo

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

Lineweaver-Burk plot (double-reciprocal)

Eadie-Hofstee plot

Hanes-Woolf Plot

Allosteric enzymes

Allosteric enzymes tend to be

multi-sub unit proteins

The reversible binding of an

allosteric modulator (here a

positive modulator M) affects

the substrate binding site

T

T

R

T

[S]

vo

Mechanism and Example of Allosteric Effect

S S

R

R

SS

RS

A

I

T[S]

vo

[S]

vo

(+)

(-) X X

X

R = Relax(active)

T = Tense(inactive)

Allosteric siteHomotropic(+)Concerted

Heterotropic(+)Sequential

Heterotropic(-)Concerted

Allosteric site

Kinetics Cooperation Models

(-)

(+)

(+)

Enzyme Inhibitors

bull Specific enzyme inhibitors regulate enzyme activity and help us

understand mechanism of enzyme action (Denaturing agents are not

inhibitors)

bull Irreversible inhibitors form covalent or very tight permanent bonds with

aa at the active site of the enzyme and render it inactive 3 classes

groupspecific reagents substrate analogs suicide inhibitors

bull Reversible inhibitors form an EI complex that can be dissociated back

to enzyme and free inhibitor 3 groups based on their mechanism of

action competitive non-competitive and uncompetitive

Enzyme Inhibition

Competitive inhibitors

bull Compete with substrate for binding to enzyme

bull E + S = ES or E + I = EI Both S and I cannot bind enzyme at the same time

bull In presence of I the equilibrium of E + S = ES is shifted to the left causing dissociation of ES

bull This can be reversed corrected by increasing [S]

bull Vmax is not changed KM is increased by (1 + IKi)

bull Eg AZT antibacterial sulfonamides the anticancer agent methotrexate etc

Competitive Inhibition

Kinetics of competitive inhibitor

Increase [S] toovercomeinhibition

Vmax attainable

Km is increased

Ki =dissociationconstant forinhibitor

V max unaltered Km increased

Non-competitive Inhibitors

bull Inhibitor binding site is distinct from substrate binding site Can bind to free enzyme E and to ES

bull E + I = EI ES + I = ESI or EI + S = ESI

bull Both EI and ESI are enzymatically inactive

bull The effective functional [E] (and [S]) is reduced

bull Reaction of unaffected ES proceeds normally

bull Inhibition cannot be reversed by increasing [S]

bull KM is not changed Vmax is decreased by (1 + IKi)

Mixed (Noncompetitive) Inhibition

Kinetics of non-competitive inhibitor

Increasing [S] cannotovercome inhibition

Less E availableV max is lowerKm remains the samefor available E

Km unaltered V max decreased

Uncompetitive Inhibitors

bull The inhibitor cannot bind to the enzyme directly but can only bind to the enzyme-substrate complex

bull ES + I = ESI

bull Both Vmax and KM are decreased by (1+IKi)

Uncompetitive Inhibition

Substrate Inhibition

Caused by high substrate concentrations

E + S ES E + PKm

rsquo k2

KS1

+

S

ES21

2

2

][][

][

][

]][[

][

]][[

Sm

m

mSi

KS

SK

SVv

ES

ESK

ES

ESSK

Substrate Inhibition

At low substrate concentrations [S]2Ks1ltlt1 and inhibition is not observed

Plot of 1v vs 1[S] gives a line Slope = Krsquo

mVm

Intercept = 1Vm

][

111

][1

SV

K

Vv

SK

Vv

m

m

m

m

m

Substrate Inhibition

At high substrate concentrations Krsquom[S]ltlt1 and

inhibition is dominant

Plot of 1v vs [S] gives a straight line Slope = 1KS1 middot Vm

Intercept = 1Vm

mSm

S

m

VK

S

Vv

KS

Vv

1

1

][11

][1

1

max][

0][

SmKKS

Sddv

1V Igt0

I=0

1Vm

-1Km -1Kmapp 1[S]

1VIgt0

I=0

1Vm

-1Km-1Kmapp 1[S]

1Vmapp

1VIgt0

I=0

1Vm

-1Km 1[S]

1Vmapp

1V

1Vm

-1Km 1[S]

Competitive Uncompetitive

Non-Competitive Substrate Inhibition

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] onlyand competes with [S]increasing [S] overcomesInhibition by [II]

[II] binds to free [E] or [ES] complex Increasing [S] cannot overcome [II] inhibition

[II] binds to [ES] complex only increasing [S] favorsthe inhibition by [II]

E + S rarr ES rarr E + P + IIdarrEII

larr

uarr

E + S rarr ES rarr E + P + + II IIdarr darrEII + S rarrEIIS

larr

uarr uarr

E + S rarr ES rarr E + P + II darr EIIS

larr

uarr

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Kmrsquo [S] mM

vo

[S] mM

vo

II II

Km [S] mM

Vmax

II

Kmrsquo

VmaxrsquoVmaxrsquo

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax amp Km decreased

II

1[S]1Km

1vo

1 Vmax

II

Two parallellines

II

Intersect at X axis

1vo

1 Vmax

1[S]1Km 1[S]1Km

1 Vmax

1vo

Intersect at Y axis

= Kmrsquo

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

Eadie-Hofstee plot

Hanes-Woolf Plot

Allosteric enzymes

Allosteric enzymes tend to be

multi-sub unit proteins

The reversible binding of an

allosteric modulator (here a

positive modulator M) affects

the substrate binding site

T

T

R

T

[S]

vo

Mechanism and Example of Allosteric Effect

S S

R

R

SS

RS

A

I

T[S]

vo

[S]

vo

(+)

(-) X X

X

R = Relax(active)

T = Tense(inactive)

Allosteric siteHomotropic(+)Concerted

Heterotropic(+)Sequential

Heterotropic(-)Concerted

Allosteric site

Kinetics Cooperation Models

(-)

(+)

(+)

Enzyme Inhibitors

bull Specific enzyme inhibitors regulate enzyme activity and help us

understand mechanism of enzyme action (Denaturing agents are not

inhibitors)

bull Irreversible inhibitors form covalent or very tight permanent bonds with

aa at the active site of the enzyme and render it inactive 3 classes

groupspecific reagents substrate analogs suicide inhibitors

bull Reversible inhibitors form an EI complex that can be dissociated back

to enzyme and free inhibitor 3 groups based on their mechanism of

action competitive non-competitive and uncompetitive

Enzyme Inhibition

Competitive inhibitors

bull Compete with substrate for binding to enzyme

bull E + S = ES or E + I = EI Both S and I cannot bind enzyme at the same time

bull In presence of I the equilibrium of E + S = ES is shifted to the left causing dissociation of ES

bull This can be reversed corrected by increasing [S]

bull Vmax is not changed KM is increased by (1 + IKi)

bull Eg AZT antibacterial sulfonamides the anticancer agent methotrexate etc

Competitive Inhibition

Kinetics of competitive inhibitor

Increase [S] toovercomeinhibition

Vmax attainable

Km is increased

Ki =dissociationconstant forinhibitor

V max unaltered Km increased

Non-competitive Inhibitors

bull Inhibitor binding site is distinct from substrate binding site Can bind to free enzyme E and to ES

bull E + I = EI ES + I = ESI or EI + S = ESI

bull Both EI and ESI are enzymatically inactive

bull The effective functional [E] (and [S]) is reduced

bull Reaction of unaffected ES proceeds normally

bull Inhibition cannot be reversed by increasing [S]

bull KM is not changed Vmax is decreased by (1 + IKi)

Mixed (Noncompetitive) Inhibition

Kinetics of non-competitive inhibitor

Increasing [S] cannotovercome inhibition

Less E availableV max is lowerKm remains the samefor available E

Km unaltered V max decreased

Uncompetitive Inhibitors

bull The inhibitor cannot bind to the enzyme directly but can only bind to the enzyme-substrate complex

bull ES + I = ESI

bull Both Vmax and KM are decreased by (1+IKi)

Uncompetitive Inhibition

Substrate Inhibition

Caused by high substrate concentrations

E + S ES E + PKm

rsquo k2

KS1

+

S

ES21

2

2

][][

][

][

]][[

][

]][[

Sm

m

mSi

KS

SK

SVv

ES

ESK

ES

ESSK

Substrate Inhibition

At low substrate concentrations [S]2Ks1ltlt1 and inhibition is not observed

Plot of 1v vs 1[S] gives a line Slope = Krsquo

mVm

Intercept = 1Vm

][

111

][1

SV

K

Vv

SK

Vv

m

m

m

m

m

Substrate Inhibition

At high substrate concentrations Krsquom[S]ltlt1 and

inhibition is dominant

Plot of 1v vs [S] gives a straight line Slope = 1KS1 middot Vm

Intercept = 1Vm

mSm

S

m

VK

S

Vv

KS

Vv

1

1

][11

][1

1

max][

0][

SmKKS

Sddv

1V Igt0

I=0

1Vm

-1Km -1Kmapp 1[S]

1VIgt0

I=0

1Vm

-1Km-1Kmapp 1[S]

1Vmapp

1VIgt0

I=0

1Vm

-1Km 1[S]

1Vmapp

1V

1Vm

-1Km 1[S]

Competitive Uncompetitive

Non-Competitive Substrate Inhibition

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] onlyand competes with [S]increasing [S] overcomesInhibition by [II]

[II] binds to free [E] or [ES] complex Increasing [S] cannot overcome [II] inhibition

[II] binds to [ES] complex only increasing [S] favorsthe inhibition by [II]

E + S rarr ES rarr E + P + IIdarrEII

larr

uarr

E + S rarr ES rarr E + P + + II IIdarr darrEII + S rarrEIIS

larr

uarr uarr

E + S rarr ES rarr E + P + II darr EIIS

larr

uarr

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Kmrsquo [S] mM

vo

[S] mM

vo

II II

Km [S] mM

Vmax

II

Kmrsquo

VmaxrsquoVmaxrsquo

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax amp Km decreased

II

1[S]1Km

1vo

1 Vmax

II

Two parallellines

II

Intersect at X axis

1vo

1 Vmax

1[S]1Km 1[S]1Km

1 Vmax

1vo

Intersect at Y axis

= Kmrsquo

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

Hanes-Woolf Plot

Allosteric enzymes

Allosteric enzymes tend to be

multi-sub unit proteins

The reversible binding of an

allosteric modulator (here a

positive modulator M) affects

the substrate binding site

T

T

R

T

[S]

vo

Mechanism and Example of Allosteric Effect

S S

R

R

SS

RS

A

I

T[S]

vo

[S]

vo

(+)

(-) X X

X

R = Relax(active)

T = Tense(inactive)

Allosteric siteHomotropic(+)Concerted

Heterotropic(+)Sequential

Heterotropic(-)Concerted

Allosteric site

Kinetics Cooperation Models

(-)

(+)

(+)

Enzyme Inhibitors

bull Specific enzyme inhibitors regulate enzyme activity and help us

understand mechanism of enzyme action (Denaturing agents are not

inhibitors)

bull Irreversible inhibitors form covalent or very tight permanent bonds with

aa at the active site of the enzyme and render it inactive 3 classes

groupspecific reagents substrate analogs suicide inhibitors

bull Reversible inhibitors form an EI complex that can be dissociated back

to enzyme and free inhibitor 3 groups based on their mechanism of

action competitive non-competitive and uncompetitive

Enzyme Inhibition

Competitive inhibitors

bull Compete with substrate for binding to enzyme

bull E + S = ES or E + I = EI Both S and I cannot bind enzyme at the same time

bull In presence of I the equilibrium of E + S = ES is shifted to the left causing dissociation of ES

bull This can be reversed corrected by increasing [S]

bull Vmax is not changed KM is increased by (1 + IKi)

bull Eg AZT antibacterial sulfonamides the anticancer agent methotrexate etc

Competitive Inhibition

Kinetics of competitive inhibitor

Increase [S] toovercomeinhibition

Vmax attainable

Km is increased

Ki =dissociationconstant forinhibitor

V max unaltered Km increased

Non-competitive Inhibitors

bull Inhibitor binding site is distinct from substrate binding site Can bind to free enzyme E and to ES

bull E + I = EI ES + I = ESI or EI + S = ESI

bull Both EI and ESI are enzymatically inactive

bull The effective functional [E] (and [S]) is reduced

bull Reaction of unaffected ES proceeds normally

bull Inhibition cannot be reversed by increasing [S]

bull KM is not changed Vmax is decreased by (1 + IKi)

Mixed (Noncompetitive) Inhibition

Kinetics of non-competitive inhibitor

Increasing [S] cannotovercome inhibition

Less E availableV max is lowerKm remains the samefor available E

Km unaltered V max decreased

Uncompetitive Inhibitors

bull The inhibitor cannot bind to the enzyme directly but can only bind to the enzyme-substrate complex

bull ES + I = ESI

bull Both Vmax and KM are decreased by (1+IKi)

Uncompetitive Inhibition

Substrate Inhibition

Caused by high substrate concentrations

E + S ES E + PKm

rsquo k2

KS1

+

S

ES21

2

2

][][

][

][

]][[

][

]][[

Sm

m

mSi

KS

SK

SVv

ES

ESK

ES

ESSK

Substrate Inhibition

At low substrate concentrations [S]2Ks1ltlt1 and inhibition is not observed

Plot of 1v vs 1[S] gives a line Slope = Krsquo

mVm

Intercept = 1Vm

][

111

][1

SV

K

Vv

SK

Vv

m

m

m

m

m

Substrate Inhibition

At high substrate concentrations Krsquom[S]ltlt1 and

inhibition is dominant

Plot of 1v vs [S] gives a straight line Slope = 1KS1 middot Vm

Intercept = 1Vm

mSm

S

m

VK

S

Vv

KS

Vv

1

1

][11

][1

1

max][

0][

SmKKS

Sddv

1V Igt0

I=0

1Vm

-1Km -1Kmapp 1[S]

1VIgt0

I=0

1Vm

-1Km-1Kmapp 1[S]

1Vmapp

1VIgt0

I=0

1Vm

-1Km 1[S]

1Vmapp

1V

1Vm

-1Km 1[S]

Competitive Uncompetitive

Non-Competitive Substrate Inhibition

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] onlyand competes with [S]increasing [S] overcomesInhibition by [II]

[II] binds to free [E] or [ES] complex Increasing [S] cannot overcome [II] inhibition

[II] binds to [ES] complex only increasing [S] favorsthe inhibition by [II]

E + S rarr ES rarr E + P + IIdarrEII

larr

uarr

E + S rarr ES rarr E + P + + II IIdarr darrEII + S rarrEIIS

larr

uarr uarr

E + S rarr ES rarr E + P + II darr EIIS

larr

uarr

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Kmrsquo [S] mM

vo

[S] mM

vo

II II

Km [S] mM

Vmax

II

Kmrsquo

VmaxrsquoVmaxrsquo

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax amp Km decreased

II

1[S]1Km

1vo

1 Vmax

II

Two parallellines

II

Intersect at X axis

1vo

1 Vmax

1[S]1Km 1[S]1Km

1 Vmax

1vo

Intersect at Y axis

= Kmrsquo

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

Allosteric enzymes

Allosteric enzymes tend to be

multi-sub unit proteins

The reversible binding of an

allosteric modulator (here a

positive modulator M) affects

the substrate binding site

T

T

R

T

[S]

vo

Mechanism and Example of Allosteric Effect

S S

R

R

SS

RS

A

I

T[S]

vo

[S]

vo

(+)

(-) X X

X

R = Relax(active)

T = Tense(inactive)

Allosteric siteHomotropic(+)Concerted

Heterotropic(+)Sequential

Heterotropic(-)Concerted

Allosteric site

Kinetics Cooperation Models

(-)

(+)

(+)

Enzyme Inhibitors

bull Specific enzyme inhibitors regulate enzyme activity and help us

understand mechanism of enzyme action (Denaturing agents are not

inhibitors)

bull Irreversible inhibitors form covalent or very tight permanent bonds with

aa at the active site of the enzyme and render it inactive 3 classes

groupspecific reagents substrate analogs suicide inhibitors

bull Reversible inhibitors form an EI complex that can be dissociated back

to enzyme and free inhibitor 3 groups based on their mechanism of

action competitive non-competitive and uncompetitive

Enzyme Inhibition

Competitive inhibitors

bull Compete with substrate for binding to enzyme

bull E + S = ES or E + I = EI Both S and I cannot bind enzyme at the same time

bull In presence of I the equilibrium of E + S = ES is shifted to the left causing dissociation of ES

bull This can be reversed corrected by increasing [S]

bull Vmax is not changed KM is increased by (1 + IKi)

bull Eg AZT antibacterial sulfonamides the anticancer agent methotrexate etc

Competitive Inhibition

Kinetics of competitive inhibitor

Increase [S] toovercomeinhibition

Vmax attainable

Km is increased

Ki =dissociationconstant forinhibitor

V max unaltered Km increased

Non-competitive Inhibitors

bull Inhibitor binding site is distinct from substrate binding site Can bind to free enzyme E and to ES

bull E + I = EI ES + I = ESI or EI + S = ESI

bull Both EI and ESI are enzymatically inactive

bull The effective functional [E] (and [S]) is reduced

bull Reaction of unaffected ES proceeds normally

bull Inhibition cannot be reversed by increasing [S]

bull KM is not changed Vmax is decreased by (1 + IKi)

Mixed (Noncompetitive) Inhibition

Kinetics of non-competitive inhibitor

Increasing [S] cannotovercome inhibition

Less E availableV max is lowerKm remains the samefor available E

Km unaltered V max decreased

Uncompetitive Inhibitors

bull The inhibitor cannot bind to the enzyme directly but can only bind to the enzyme-substrate complex

bull ES + I = ESI

bull Both Vmax and KM are decreased by (1+IKi)

Uncompetitive Inhibition

Substrate Inhibition

Caused by high substrate concentrations

E + S ES E + PKm

rsquo k2

KS1

+

S

ES21

2

2

][][

][

][

]][[

][

]][[

Sm

m

mSi

KS

SK

SVv

ES

ESK

ES

ESSK

Substrate Inhibition

At low substrate concentrations [S]2Ks1ltlt1 and inhibition is not observed

Plot of 1v vs 1[S] gives a line Slope = Krsquo

mVm

Intercept = 1Vm

][

111

][1

SV

K

Vv

SK

Vv

m

m

m

m

m

Substrate Inhibition

At high substrate concentrations Krsquom[S]ltlt1 and

inhibition is dominant

Plot of 1v vs [S] gives a straight line Slope = 1KS1 middot Vm

Intercept = 1Vm

mSm

S

m

VK

S

Vv

KS

Vv

1

1

][11

][1

1

max][

0][

SmKKS

Sddv

1V Igt0

I=0

1Vm

-1Km -1Kmapp 1[S]

1VIgt0

I=0

1Vm

-1Km-1Kmapp 1[S]

1Vmapp

1VIgt0

I=0

1Vm

-1Km 1[S]

1Vmapp

1V

1Vm

-1Km 1[S]

Competitive Uncompetitive

Non-Competitive Substrate Inhibition

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] onlyand competes with [S]increasing [S] overcomesInhibition by [II]

[II] binds to free [E] or [ES] complex Increasing [S] cannot overcome [II] inhibition

[II] binds to [ES] complex only increasing [S] favorsthe inhibition by [II]

E + S rarr ES rarr E + P + IIdarrEII

larr

uarr

E + S rarr ES rarr E + P + + II IIdarr darrEII + S rarrEIIS

larr

uarr uarr

E + S rarr ES rarr E + P + II darr EIIS

larr

uarr

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Kmrsquo [S] mM

vo

[S] mM

vo

II II

Km [S] mM

Vmax

II

Kmrsquo

VmaxrsquoVmaxrsquo

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax amp Km decreased

II

1[S]1Km

1vo

1 Vmax

II

Two parallellines

II

Intersect at X axis

1vo

1 Vmax

1[S]1Km 1[S]1Km

1 Vmax

1vo

Intersect at Y axis

= Kmrsquo

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

T

T

R

T

[S]

vo

Mechanism and Example of Allosteric Effect

S S

R

R

SS

RS

A

I

T[S]

vo

[S]

vo

(+)

(-) X X

X

R = Relax(active)

T = Tense(inactive)

Allosteric siteHomotropic(+)Concerted

Heterotropic(+)Sequential

Heterotropic(-)Concerted

Allosteric site

Kinetics Cooperation Models

(-)

(+)

(+)

Enzyme Inhibitors

bull Specific enzyme inhibitors regulate enzyme activity and help us

understand mechanism of enzyme action (Denaturing agents are not

inhibitors)

bull Irreversible inhibitors form covalent or very tight permanent bonds with

aa at the active site of the enzyme and render it inactive 3 classes

groupspecific reagents substrate analogs suicide inhibitors

bull Reversible inhibitors form an EI complex that can be dissociated back

to enzyme and free inhibitor 3 groups based on their mechanism of

action competitive non-competitive and uncompetitive

Enzyme Inhibition

Competitive inhibitors

bull Compete with substrate for binding to enzyme

bull E + S = ES or E + I = EI Both S and I cannot bind enzyme at the same time

bull In presence of I the equilibrium of E + S = ES is shifted to the left causing dissociation of ES

bull This can be reversed corrected by increasing [S]

bull Vmax is not changed KM is increased by (1 + IKi)

bull Eg AZT antibacterial sulfonamides the anticancer agent methotrexate etc

Competitive Inhibition

Kinetics of competitive inhibitor

Increase [S] toovercomeinhibition

Vmax attainable

Km is increased

Ki =dissociationconstant forinhibitor

V max unaltered Km increased

Non-competitive Inhibitors

bull Inhibitor binding site is distinct from substrate binding site Can bind to free enzyme E and to ES

bull E + I = EI ES + I = ESI or EI + S = ESI

bull Both EI and ESI are enzymatically inactive

bull The effective functional [E] (and [S]) is reduced

bull Reaction of unaffected ES proceeds normally

bull Inhibition cannot be reversed by increasing [S]

bull KM is not changed Vmax is decreased by (1 + IKi)

Mixed (Noncompetitive) Inhibition

Kinetics of non-competitive inhibitor

Increasing [S] cannotovercome inhibition

Less E availableV max is lowerKm remains the samefor available E

Km unaltered V max decreased

Uncompetitive Inhibitors

bull The inhibitor cannot bind to the enzyme directly but can only bind to the enzyme-substrate complex

bull ES + I = ESI

bull Both Vmax and KM are decreased by (1+IKi)

Uncompetitive Inhibition

Substrate Inhibition

Caused by high substrate concentrations

E + S ES E + PKm

rsquo k2

KS1

+

S

ES21

2

2

][][

][

][

]][[

][

]][[

Sm

m

mSi

KS

SK

SVv

ES

ESK

ES

ESSK

Substrate Inhibition

At low substrate concentrations [S]2Ks1ltlt1 and inhibition is not observed

Plot of 1v vs 1[S] gives a line Slope = Krsquo

mVm

Intercept = 1Vm

][

111

][1

SV

K

Vv

SK

Vv

m

m

m

m

m

Substrate Inhibition

At high substrate concentrations Krsquom[S]ltlt1 and

inhibition is dominant

Plot of 1v vs [S] gives a straight line Slope = 1KS1 middot Vm

Intercept = 1Vm

mSm

S

m

VK

S

Vv

KS

Vv

1

1

][11

][1

1

max][

0][

SmKKS

Sddv

1V Igt0

I=0

1Vm

-1Km -1Kmapp 1[S]

1VIgt0

I=0

1Vm

-1Km-1Kmapp 1[S]

1Vmapp

1VIgt0

I=0

1Vm

-1Km 1[S]

1Vmapp

1V

1Vm

-1Km 1[S]

Competitive Uncompetitive

Non-Competitive Substrate Inhibition

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] onlyand competes with [S]increasing [S] overcomesInhibition by [II]

[II] binds to free [E] or [ES] complex Increasing [S] cannot overcome [II] inhibition

[II] binds to [ES] complex only increasing [S] favorsthe inhibition by [II]

E + S rarr ES rarr E + P + IIdarrEII

larr

uarr

E + S rarr ES rarr E + P + + II IIdarr darrEII + S rarrEIIS

larr

uarr uarr

E + S rarr ES rarr E + P + II darr EIIS

larr

uarr

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Kmrsquo [S] mM

vo

[S] mM

vo

II II

Km [S] mM

Vmax

II

Kmrsquo

VmaxrsquoVmaxrsquo

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax amp Km decreased

II

1[S]1Km

1vo

1 Vmax

II

Two parallellines

II

Intersect at X axis

1vo

1 Vmax

1[S]1Km 1[S]1Km

1 Vmax

1vo

Intersect at Y axis

= Kmrsquo

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

Enzyme Inhibitors

bull Specific enzyme inhibitors regulate enzyme activity and help us

understand mechanism of enzyme action (Denaturing agents are not

inhibitors)

bull Irreversible inhibitors form covalent or very tight permanent bonds with

aa at the active site of the enzyme and render it inactive 3 classes

groupspecific reagents substrate analogs suicide inhibitors

bull Reversible inhibitors form an EI complex that can be dissociated back

to enzyme and free inhibitor 3 groups based on their mechanism of

action competitive non-competitive and uncompetitive

Enzyme Inhibition

Competitive inhibitors

bull Compete with substrate for binding to enzyme

bull E + S = ES or E + I = EI Both S and I cannot bind enzyme at the same time

bull In presence of I the equilibrium of E + S = ES is shifted to the left causing dissociation of ES

bull This can be reversed corrected by increasing [S]

bull Vmax is not changed KM is increased by (1 + IKi)

bull Eg AZT antibacterial sulfonamides the anticancer agent methotrexate etc

Competitive Inhibition

Kinetics of competitive inhibitor

Increase [S] toovercomeinhibition

Vmax attainable

Km is increased

Ki =dissociationconstant forinhibitor

V max unaltered Km increased

Non-competitive Inhibitors

bull Inhibitor binding site is distinct from substrate binding site Can bind to free enzyme E and to ES

bull E + I = EI ES + I = ESI or EI + S = ESI

bull Both EI and ESI are enzymatically inactive

bull The effective functional [E] (and [S]) is reduced

bull Reaction of unaffected ES proceeds normally

bull Inhibition cannot be reversed by increasing [S]

bull KM is not changed Vmax is decreased by (1 + IKi)

Mixed (Noncompetitive) Inhibition

Kinetics of non-competitive inhibitor

Increasing [S] cannotovercome inhibition

Less E availableV max is lowerKm remains the samefor available E

Km unaltered V max decreased

Uncompetitive Inhibitors

bull The inhibitor cannot bind to the enzyme directly but can only bind to the enzyme-substrate complex

bull ES + I = ESI

bull Both Vmax and KM are decreased by (1+IKi)

Uncompetitive Inhibition

Substrate Inhibition

Caused by high substrate concentrations

E + S ES E + PKm

rsquo k2

KS1

+

S

ES21

2

2

][][

][

][

]][[

][

]][[

Sm

m

mSi

KS

SK

SVv

ES

ESK

ES

ESSK

Substrate Inhibition

At low substrate concentrations [S]2Ks1ltlt1 and inhibition is not observed

Plot of 1v vs 1[S] gives a line Slope = Krsquo

mVm

Intercept = 1Vm

][

111

][1

SV

K

Vv

SK

Vv

m

m

m

m

m

Substrate Inhibition

At high substrate concentrations Krsquom[S]ltlt1 and

inhibition is dominant

Plot of 1v vs [S] gives a straight line Slope = 1KS1 middot Vm

Intercept = 1Vm

mSm

S

m

VK

S

Vv

KS

Vv

1

1

][11

][1

1

max][

0][

SmKKS

Sddv

1V Igt0

I=0

1Vm

-1Km -1Kmapp 1[S]

1VIgt0

I=0

1Vm

-1Km-1Kmapp 1[S]

1Vmapp

1VIgt0

I=0

1Vm

-1Km 1[S]

1Vmapp

1V

1Vm

-1Km 1[S]

Competitive Uncompetitive

Non-Competitive Substrate Inhibition

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] onlyand competes with [S]increasing [S] overcomesInhibition by [II]

[II] binds to free [E] or [ES] complex Increasing [S] cannot overcome [II] inhibition

[II] binds to [ES] complex only increasing [S] favorsthe inhibition by [II]

E + S rarr ES rarr E + P + IIdarrEII

larr

uarr

E + S rarr ES rarr E + P + + II IIdarr darrEII + S rarrEIIS

larr

uarr uarr

E + S rarr ES rarr E + P + II darr EIIS

larr

uarr

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Kmrsquo [S] mM

vo

[S] mM

vo

II II

Km [S] mM

Vmax

II

Kmrsquo

VmaxrsquoVmaxrsquo

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax amp Km decreased

II

1[S]1Km

1vo

1 Vmax

II

Two parallellines

II

Intersect at X axis

1vo

1 Vmax

1[S]1Km 1[S]1Km

1 Vmax

1vo

Intersect at Y axis

= Kmrsquo

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

Enzyme Inhibition

Competitive inhibitors

bull Compete with substrate for binding to enzyme

bull E + S = ES or E + I = EI Both S and I cannot bind enzyme at the same time

bull In presence of I the equilibrium of E + S = ES is shifted to the left causing dissociation of ES

bull This can be reversed corrected by increasing [S]

bull Vmax is not changed KM is increased by (1 + IKi)

bull Eg AZT antibacterial sulfonamides the anticancer agent methotrexate etc

Competitive Inhibition

Kinetics of competitive inhibitor

Increase [S] toovercomeinhibition

Vmax attainable

Km is increased

Ki =dissociationconstant forinhibitor

V max unaltered Km increased

Non-competitive Inhibitors

bull Inhibitor binding site is distinct from substrate binding site Can bind to free enzyme E and to ES

bull E + I = EI ES + I = ESI or EI + S = ESI

bull Both EI and ESI are enzymatically inactive

bull The effective functional [E] (and [S]) is reduced

bull Reaction of unaffected ES proceeds normally

bull Inhibition cannot be reversed by increasing [S]

bull KM is not changed Vmax is decreased by (1 + IKi)

Mixed (Noncompetitive) Inhibition

Kinetics of non-competitive inhibitor

Increasing [S] cannotovercome inhibition

Less E availableV max is lowerKm remains the samefor available E

Km unaltered V max decreased

Uncompetitive Inhibitors

bull The inhibitor cannot bind to the enzyme directly but can only bind to the enzyme-substrate complex

bull ES + I = ESI

bull Both Vmax and KM are decreased by (1+IKi)

Uncompetitive Inhibition

Substrate Inhibition

Caused by high substrate concentrations

E + S ES E + PKm

rsquo k2

KS1

+

S

ES21

2

2

][][

][

][

]][[

][

]][[

Sm

m

mSi

KS

SK

SVv

ES

ESK

ES

ESSK

Substrate Inhibition

At low substrate concentrations [S]2Ks1ltlt1 and inhibition is not observed

Plot of 1v vs 1[S] gives a line Slope = Krsquo

mVm

Intercept = 1Vm

][

111

][1

SV

K

Vv

SK

Vv

m

m

m

m

m

Substrate Inhibition

At high substrate concentrations Krsquom[S]ltlt1 and

inhibition is dominant

Plot of 1v vs [S] gives a straight line Slope = 1KS1 middot Vm

Intercept = 1Vm

mSm

S

m

VK

S

Vv

KS

Vv

1

1

][11

][1

1

max][

0][

SmKKS

Sddv

1V Igt0

I=0

1Vm

-1Km -1Kmapp 1[S]

1VIgt0

I=0

1Vm

-1Km-1Kmapp 1[S]

1Vmapp

1VIgt0

I=0

1Vm

-1Km 1[S]

1Vmapp

1V

1Vm

-1Km 1[S]

Competitive Uncompetitive

Non-Competitive Substrate Inhibition

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] onlyand competes with [S]increasing [S] overcomesInhibition by [II]

[II] binds to free [E] or [ES] complex Increasing [S] cannot overcome [II] inhibition

[II] binds to [ES] complex only increasing [S] favorsthe inhibition by [II]

E + S rarr ES rarr E + P + IIdarrEII

larr

uarr

E + S rarr ES rarr E + P + + II IIdarr darrEII + S rarrEIIS

larr

uarr uarr

E + S rarr ES rarr E + P + II darr EIIS

larr

uarr

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Kmrsquo [S] mM

vo

[S] mM

vo

II II

Km [S] mM

Vmax

II

Kmrsquo

VmaxrsquoVmaxrsquo

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax amp Km decreased

II

1[S]1Km

1vo

1 Vmax

II

Two parallellines

II

Intersect at X axis

1vo

1 Vmax

1[S]1Km 1[S]1Km

1 Vmax

1vo

Intersect at Y axis

= Kmrsquo

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

Competitive inhibitors

bull Compete with substrate for binding to enzyme

bull E + S = ES or E + I = EI Both S and I cannot bind enzyme at the same time

bull In presence of I the equilibrium of E + S = ES is shifted to the left causing dissociation of ES

bull This can be reversed corrected by increasing [S]

bull Vmax is not changed KM is increased by (1 + IKi)

bull Eg AZT antibacterial sulfonamides the anticancer agent methotrexate etc

Competitive Inhibition

Kinetics of competitive inhibitor

Increase [S] toovercomeinhibition

Vmax attainable

Km is increased

Ki =dissociationconstant forinhibitor

V max unaltered Km increased

Non-competitive Inhibitors

bull Inhibitor binding site is distinct from substrate binding site Can bind to free enzyme E and to ES

bull E + I = EI ES + I = ESI or EI + S = ESI

bull Both EI and ESI are enzymatically inactive

bull The effective functional [E] (and [S]) is reduced

bull Reaction of unaffected ES proceeds normally

bull Inhibition cannot be reversed by increasing [S]

bull KM is not changed Vmax is decreased by (1 + IKi)

Mixed (Noncompetitive) Inhibition

Kinetics of non-competitive inhibitor

Increasing [S] cannotovercome inhibition

Less E availableV max is lowerKm remains the samefor available E

Km unaltered V max decreased

Uncompetitive Inhibitors

bull The inhibitor cannot bind to the enzyme directly but can only bind to the enzyme-substrate complex

bull ES + I = ESI

bull Both Vmax and KM are decreased by (1+IKi)

Uncompetitive Inhibition

Substrate Inhibition

Caused by high substrate concentrations

E + S ES E + PKm

rsquo k2

KS1

+

S

ES21

2

2

][][

][

][

]][[

][

]][[

Sm

m

mSi

KS

SK

SVv

ES

ESK

ES

ESSK

Substrate Inhibition

At low substrate concentrations [S]2Ks1ltlt1 and inhibition is not observed

Plot of 1v vs 1[S] gives a line Slope = Krsquo

mVm

Intercept = 1Vm

][

111

][1

SV

K

Vv

SK

Vv

m

m

m

m

m

Substrate Inhibition

At high substrate concentrations Krsquom[S]ltlt1 and

inhibition is dominant

Plot of 1v vs [S] gives a straight line Slope = 1KS1 middot Vm

Intercept = 1Vm

mSm

S

m

VK

S

Vv

KS

Vv

1

1

][11

][1

1

max][

0][

SmKKS

Sddv

1V Igt0

I=0

1Vm

-1Km -1Kmapp 1[S]

1VIgt0

I=0

1Vm

-1Km-1Kmapp 1[S]

1Vmapp

1VIgt0

I=0

1Vm

-1Km 1[S]

1Vmapp

1V

1Vm

-1Km 1[S]

Competitive Uncompetitive

Non-Competitive Substrate Inhibition

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] onlyand competes with [S]increasing [S] overcomesInhibition by [II]

[II] binds to free [E] or [ES] complex Increasing [S] cannot overcome [II] inhibition

[II] binds to [ES] complex only increasing [S] favorsthe inhibition by [II]

E + S rarr ES rarr E + P + IIdarrEII

larr

uarr

E + S rarr ES rarr E + P + + II IIdarr darrEII + S rarrEIIS

larr

uarr uarr

E + S rarr ES rarr E + P + II darr EIIS

larr

uarr

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Kmrsquo [S] mM

vo

[S] mM

vo

II II

Km [S] mM

Vmax

II

Kmrsquo

VmaxrsquoVmaxrsquo

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax amp Km decreased

II

1[S]1Km

1vo

1 Vmax

II

Two parallellines

II

Intersect at X axis

1vo

1 Vmax

1[S]1Km 1[S]1Km

1 Vmax

1vo

Intersect at Y axis

= Kmrsquo

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

Competitive Inhibition

Kinetics of competitive inhibitor

Increase [S] toovercomeinhibition

Vmax attainable

Km is increased

Ki =dissociationconstant forinhibitor

V max unaltered Km increased

Non-competitive Inhibitors

bull Inhibitor binding site is distinct from substrate binding site Can bind to free enzyme E and to ES

bull E + I = EI ES + I = ESI or EI + S = ESI

bull Both EI and ESI are enzymatically inactive

bull The effective functional [E] (and [S]) is reduced

bull Reaction of unaffected ES proceeds normally

bull Inhibition cannot be reversed by increasing [S]

bull KM is not changed Vmax is decreased by (1 + IKi)

Mixed (Noncompetitive) Inhibition

Kinetics of non-competitive inhibitor

Increasing [S] cannotovercome inhibition

Less E availableV max is lowerKm remains the samefor available E

Km unaltered V max decreased

Uncompetitive Inhibitors

bull The inhibitor cannot bind to the enzyme directly but can only bind to the enzyme-substrate complex

bull ES + I = ESI

bull Both Vmax and KM are decreased by (1+IKi)

Uncompetitive Inhibition

Substrate Inhibition

Caused by high substrate concentrations

E + S ES E + PKm

rsquo k2

KS1

+

S

ES21

2

2

][][

][

][

]][[

][

]][[

Sm

m

mSi

KS

SK

SVv

ES

ESK

ES

ESSK

Substrate Inhibition

At low substrate concentrations [S]2Ks1ltlt1 and inhibition is not observed

Plot of 1v vs 1[S] gives a line Slope = Krsquo

mVm

Intercept = 1Vm

][

111

][1

SV

K

Vv

SK

Vv

m

m

m

m

m

Substrate Inhibition

At high substrate concentrations Krsquom[S]ltlt1 and

inhibition is dominant

Plot of 1v vs [S] gives a straight line Slope = 1KS1 middot Vm

Intercept = 1Vm

mSm

S

m

VK

S

Vv

KS

Vv

1

1

][11

][1

1

max][

0][

SmKKS

Sddv

1V Igt0

I=0

1Vm

-1Km -1Kmapp 1[S]

1VIgt0

I=0

1Vm

-1Km-1Kmapp 1[S]

1Vmapp

1VIgt0

I=0

1Vm

-1Km 1[S]

1Vmapp

1V

1Vm

-1Km 1[S]

Competitive Uncompetitive

Non-Competitive Substrate Inhibition

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] onlyand competes with [S]increasing [S] overcomesInhibition by [II]

[II] binds to free [E] or [ES] complex Increasing [S] cannot overcome [II] inhibition

[II] binds to [ES] complex only increasing [S] favorsthe inhibition by [II]

E + S rarr ES rarr E + P + IIdarrEII

larr

uarr

E + S rarr ES rarr E + P + + II IIdarr darrEII + S rarrEIIS

larr

uarr uarr

E + S rarr ES rarr E + P + II darr EIIS

larr

uarr

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Kmrsquo [S] mM

vo

[S] mM

vo

II II

Km [S] mM

Vmax

II

Kmrsquo

VmaxrsquoVmaxrsquo

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax amp Km decreased

II

1[S]1Km

1vo

1 Vmax

II

Two parallellines

II

Intersect at X axis

1vo

1 Vmax

1[S]1Km 1[S]1Km

1 Vmax

1vo

Intersect at Y axis

= Kmrsquo

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

Kinetics of competitive inhibitor

Increase [S] toovercomeinhibition

Vmax attainable

Km is increased

Ki =dissociationconstant forinhibitor

V max unaltered Km increased

Non-competitive Inhibitors

bull Inhibitor binding site is distinct from substrate binding site Can bind to free enzyme E and to ES

bull E + I = EI ES + I = ESI or EI + S = ESI

bull Both EI and ESI are enzymatically inactive

bull The effective functional [E] (and [S]) is reduced

bull Reaction of unaffected ES proceeds normally

bull Inhibition cannot be reversed by increasing [S]

bull KM is not changed Vmax is decreased by (1 + IKi)

Mixed (Noncompetitive) Inhibition

Kinetics of non-competitive inhibitor

Increasing [S] cannotovercome inhibition

Less E availableV max is lowerKm remains the samefor available E

Km unaltered V max decreased

Uncompetitive Inhibitors

bull The inhibitor cannot bind to the enzyme directly but can only bind to the enzyme-substrate complex

bull ES + I = ESI

bull Both Vmax and KM are decreased by (1+IKi)

Uncompetitive Inhibition

Substrate Inhibition

Caused by high substrate concentrations

E + S ES E + PKm

rsquo k2

KS1

+

S

ES21

2

2

][][

][

][

]][[

][

]][[

Sm

m

mSi

KS

SK

SVv

ES

ESK

ES

ESSK

Substrate Inhibition

At low substrate concentrations [S]2Ks1ltlt1 and inhibition is not observed

Plot of 1v vs 1[S] gives a line Slope = Krsquo

mVm

Intercept = 1Vm

][

111

][1

SV

K

Vv

SK

Vv

m

m

m

m

m

Substrate Inhibition

At high substrate concentrations Krsquom[S]ltlt1 and

inhibition is dominant

Plot of 1v vs [S] gives a straight line Slope = 1KS1 middot Vm

Intercept = 1Vm

mSm

S

m

VK

S

Vv

KS

Vv

1

1

][11

][1

1

max][

0][

SmKKS

Sddv

1V Igt0

I=0

1Vm

-1Km -1Kmapp 1[S]

1VIgt0

I=0

1Vm

-1Km-1Kmapp 1[S]

1Vmapp

1VIgt0

I=0

1Vm

-1Km 1[S]

1Vmapp

1V

1Vm

-1Km 1[S]

Competitive Uncompetitive

Non-Competitive Substrate Inhibition

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] onlyand competes with [S]increasing [S] overcomesInhibition by [II]

[II] binds to free [E] or [ES] complex Increasing [S] cannot overcome [II] inhibition

[II] binds to [ES] complex only increasing [S] favorsthe inhibition by [II]

E + S rarr ES rarr E + P + IIdarrEII

larr

uarr

E + S rarr ES rarr E + P + + II IIdarr darrEII + S rarrEIIS

larr

uarr uarr

E + S rarr ES rarr E + P + II darr EIIS

larr

uarr

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Kmrsquo [S] mM

vo

[S] mM

vo

II II

Km [S] mM

Vmax

II

Kmrsquo

VmaxrsquoVmaxrsquo

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax amp Km decreased

II

1[S]1Km

1vo

1 Vmax

II

Two parallellines

II

Intersect at X axis

1vo

1 Vmax

1[S]1Km 1[S]1Km

1 Vmax

1vo

Intersect at Y axis

= Kmrsquo

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

V max unaltered Km increased

Non-competitive Inhibitors

bull Inhibitor binding site is distinct from substrate binding site Can bind to free enzyme E and to ES

bull E + I = EI ES + I = ESI or EI + S = ESI

bull Both EI and ESI are enzymatically inactive

bull The effective functional [E] (and [S]) is reduced

bull Reaction of unaffected ES proceeds normally

bull Inhibition cannot be reversed by increasing [S]

bull KM is not changed Vmax is decreased by (1 + IKi)

Mixed (Noncompetitive) Inhibition

Kinetics of non-competitive inhibitor

Increasing [S] cannotovercome inhibition

Less E availableV max is lowerKm remains the samefor available E

Km unaltered V max decreased

Uncompetitive Inhibitors

bull The inhibitor cannot bind to the enzyme directly but can only bind to the enzyme-substrate complex

bull ES + I = ESI

bull Both Vmax and KM are decreased by (1+IKi)

Uncompetitive Inhibition

Substrate Inhibition

Caused by high substrate concentrations

E + S ES E + PKm

rsquo k2

KS1

+

S

ES21

2

2

][][

][

][

]][[

][

]][[

Sm

m

mSi

KS

SK

SVv

ES

ESK

ES

ESSK

Substrate Inhibition

At low substrate concentrations [S]2Ks1ltlt1 and inhibition is not observed

Plot of 1v vs 1[S] gives a line Slope = Krsquo

mVm

Intercept = 1Vm

][

111

][1

SV

K

Vv

SK

Vv

m

m

m

m

m

Substrate Inhibition

At high substrate concentrations Krsquom[S]ltlt1 and

inhibition is dominant

Plot of 1v vs [S] gives a straight line Slope = 1KS1 middot Vm

Intercept = 1Vm

mSm

S

m

VK

S

Vv

KS

Vv

1

1

][11

][1

1

max][

0][

SmKKS

Sddv

1V Igt0

I=0

1Vm

-1Km -1Kmapp 1[S]

1VIgt0

I=0

1Vm

-1Km-1Kmapp 1[S]

1Vmapp

1VIgt0

I=0

1Vm

-1Km 1[S]

1Vmapp

1V

1Vm

-1Km 1[S]

Competitive Uncompetitive

Non-Competitive Substrate Inhibition

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] onlyand competes with [S]increasing [S] overcomesInhibition by [II]

[II] binds to free [E] or [ES] complex Increasing [S] cannot overcome [II] inhibition

[II] binds to [ES] complex only increasing [S] favorsthe inhibition by [II]

E + S rarr ES rarr E + P + IIdarrEII

larr

uarr

E + S rarr ES rarr E + P + + II IIdarr darrEII + S rarrEIIS

larr

uarr uarr

E + S rarr ES rarr E + P + II darr EIIS

larr

uarr

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Kmrsquo [S] mM

vo

[S] mM

vo

II II

Km [S] mM

Vmax

II

Kmrsquo

VmaxrsquoVmaxrsquo

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax amp Km decreased

II

1[S]1Km

1vo

1 Vmax

II

Two parallellines

II

Intersect at X axis

1vo

1 Vmax

1[S]1Km 1[S]1Km

1 Vmax

1vo

Intersect at Y axis

= Kmrsquo

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

Non-competitive Inhibitors

bull Inhibitor binding site is distinct from substrate binding site Can bind to free enzyme E and to ES

bull E + I = EI ES + I = ESI or EI + S = ESI

bull Both EI and ESI are enzymatically inactive

bull The effective functional [E] (and [S]) is reduced

bull Reaction of unaffected ES proceeds normally

bull Inhibition cannot be reversed by increasing [S]

bull KM is not changed Vmax is decreased by (1 + IKi)

Mixed (Noncompetitive) Inhibition

Kinetics of non-competitive inhibitor

Increasing [S] cannotovercome inhibition

Less E availableV max is lowerKm remains the samefor available E

Km unaltered V max decreased

Uncompetitive Inhibitors

bull The inhibitor cannot bind to the enzyme directly but can only bind to the enzyme-substrate complex

bull ES + I = ESI

bull Both Vmax and KM are decreased by (1+IKi)

Uncompetitive Inhibition

Substrate Inhibition

Caused by high substrate concentrations

E + S ES E + PKm

rsquo k2

KS1

+

S

ES21

2

2

][][

][

][

]][[

][

]][[

Sm

m

mSi

KS

SK

SVv

ES

ESK

ES

ESSK

Substrate Inhibition

At low substrate concentrations [S]2Ks1ltlt1 and inhibition is not observed

Plot of 1v vs 1[S] gives a line Slope = Krsquo

mVm

Intercept = 1Vm

][

111

][1

SV

K

Vv

SK

Vv

m

m

m

m

m

Substrate Inhibition

At high substrate concentrations Krsquom[S]ltlt1 and

inhibition is dominant

Plot of 1v vs [S] gives a straight line Slope = 1KS1 middot Vm

Intercept = 1Vm

mSm

S

m

VK

S

Vv

KS

Vv

1

1

][11

][1

1

max][

0][

SmKKS

Sddv

1V Igt0

I=0

1Vm

-1Km -1Kmapp 1[S]

1VIgt0

I=0

1Vm

-1Km-1Kmapp 1[S]

1Vmapp

1VIgt0

I=0

1Vm

-1Km 1[S]

1Vmapp

1V

1Vm

-1Km 1[S]

Competitive Uncompetitive

Non-Competitive Substrate Inhibition

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] onlyand competes with [S]increasing [S] overcomesInhibition by [II]

[II] binds to free [E] or [ES] complex Increasing [S] cannot overcome [II] inhibition

[II] binds to [ES] complex only increasing [S] favorsthe inhibition by [II]

E + S rarr ES rarr E + P + IIdarrEII

larr

uarr

E + S rarr ES rarr E + P + + II IIdarr darrEII + S rarrEIIS

larr

uarr uarr

E + S rarr ES rarr E + P + II darr EIIS

larr

uarr

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Kmrsquo [S] mM

vo

[S] mM

vo

II II

Km [S] mM

Vmax

II

Kmrsquo

VmaxrsquoVmaxrsquo

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax amp Km decreased

II

1[S]1Km

1vo

1 Vmax

II

Two parallellines

II

Intersect at X axis

1vo

1 Vmax

1[S]1Km 1[S]1Km

1 Vmax

1vo

Intersect at Y axis

= Kmrsquo

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

Mixed (Noncompetitive) Inhibition

Kinetics of non-competitive inhibitor

Increasing [S] cannotovercome inhibition

Less E availableV max is lowerKm remains the samefor available E

Km unaltered V max decreased

Uncompetitive Inhibitors

bull The inhibitor cannot bind to the enzyme directly but can only bind to the enzyme-substrate complex

bull ES + I = ESI

bull Both Vmax and KM are decreased by (1+IKi)

Uncompetitive Inhibition

Substrate Inhibition

Caused by high substrate concentrations

E + S ES E + PKm

rsquo k2

KS1

+

S

ES21

2

2

][][

][

][

]][[

][

]][[

Sm

m

mSi

KS

SK

SVv

ES

ESK

ES

ESSK

Substrate Inhibition

At low substrate concentrations [S]2Ks1ltlt1 and inhibition is not observed

Plot of 1v vs 1[S] gives a line Slope = Krsquo

mVm

Intercept = 1Vm

][

111

][1

SV

K

Vv

SK

Vv

m

m

m

m

m

Substrate Inhibition

At high substrate concentrations Krsquom[S]ltlt1 and

inhibition is dominant

Plot of 1v vs [S] gives a straight line Slope = 1KS1 middot Vm

Intercept = 1Vm

mSm

S

m

VK

S

Vv

KS

Vv

1

1

][11

][1

1

max][

0][

SmKKS

Sddv

1V Igt0

I=0

1Vm

-1Km -1Kmapp 1[S]

1VIgt0

I=0

1Vm

-1Km-1Kmapp 1[S]

1Vmapp

1VIgt0

I=0

1Vm

-1Km 1[S]

1Vmapp

1V

1Vm

-1Km 1[S]

Competitive Uncompetitive

Non-Competitive Substrate Inhibition

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] onlyand competes with [S]increasing [S] overcomesInhibition by [II]

[II] binds to free [E] or [ES] complex Increasing [S] cannot overcome [II] inhibition

[II] binds to [ES] complex only increasing [S] favorsthe inhibition by [II]

E + S rarr ES rarr E + P + IIdarrEII

larr

uarr

E + S rarr ES rarr E + P + + II IIdarr darrEII + S rarrEIIS

larr

uarr uarr

E + S rarr ES rarr E + P + II darr EIIS

larr

uarr

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Kmrsquo [S] mM

vo

[S] mM

vo

II II

Km [S] mM

Vmax

II

Kmrsquo

VmaxrsquoVmaxrsquo

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax amp Km decreased

II

1[S]1Km

1vo

1 Vmax

II

Two parallellines

II

Intersect at X axis

1vo

1 Vmax

1[S]1Km 1[S]1Km

1 Vmax

1vo

Intersect at Y axis

= Kmrsquo

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

Kinetics of non-competitive inhibitor

Increasing [S] cannotovercome inhibition

Less E availableV max is lowerKm remains the samefor available E

Km unaltered V max decreased

Uncompetitive Inhibitors

bull The inhibitor cannot bind to the enzyme directly but can only bind to the enzyme-substrate complex

bull ES + I = ESI

bull Both Vmax and KM are decreased by (1+IKi)

Uncompetitive Inhibition

Substrate Inhibition

Caused by high substrate concentrations

E + S ES E + PKm

rsquo k2

KS1

+

S

ES21

2

2

][][

][

][

]][[

][

]][[

Sm

m

mSi

KS

SK

SVv

ES

ESK

ES

ESSK

Substrate Inhibition

At low substrate concentrations [S]2Ks1ltlt1 and inhibition is not observed

Plot of 1v vs 1[S] gives a line Slope = Krsquo

mVm

Intercept = 1Vm

][

111

][1

SV

K

Vv

SK

Vv

m

m

m

m

m

Substrate Inhibition

At high substrate concentrations Krsquom[S]ltlt1 and

inhibition is dominant

Plot of 1v vs [S] gives a straight line Slope = 1KS1 middot Vm

Intercept = 1Vm

mSm

S

m

VK

S

Vv

KS

Vv

1

1

][11

][1

1

max][

0][

SmKKS

Sddv

1V Igt0

I=0

1Vm

-1Km -1Kmapp 1[S]

1VIgt0

I=0

1Vm

-1Km-1Kmapp 1[S]

1Vmapp

1VIgt0

I=0

1Vm

-1Km 1[S]

1Vmapp

1V

1Vm

-1Km 1[S]

Competitive Uncompetitive

Non-Competitive Substrate Inhibition

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] onlyand competes with [S]increasing [S] overcomesInhibition by [II]

[II] binds to free [E] or [ES] complex Increasing [S] cannot overcome [II] inhibition

[II] binds to [ES] complex only increasing [S] favorsthe inhibition by [II]

E + S rarr ES rarr E + P + IIdarrEII

larr

uarr

E + S rarr ES rarr E + P + + II IIdarr darrEII + S rarrEIIS

larr

uarr uarr

E + S rarr ES rarr E + P + II darr EIIS

larr

uarr

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Kmrsquo [S] mM

vo

[S] mM

vo

II II

Km [S] mM

Vmax

II

Kmrsquo

VmaxrsquoVmaxrsquo

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax amp Km decreased

II

1[S]1Km

1vo

1 Vmax

II

Two parallellines

II

Intersect at X axis

1vo

1 Vmax

1[S]1Km 1[S]1Km

1 Vmax

1vo

Intersect at Y axis

= Kmrsquo

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

Km unaltered V max decreased

Uncompetitive Inhibitors

bull The inhibitor cannot bind to the enzyme directly but can only bind to the enzyme-substrate complex

bull ES + I = ESI

bull Both Vmax and KM are decreased by (1+IKi)

Uncompetitive Inhibition

Substrate Inhibition

Caused by high substrate concentrations

E + S ES E + PKm

rsquo k2

KS1

+

S

ES21

2

2

][][

][

][

]][[

][

]][[

Sm

m

mSi

KS

SK

SVv

ES

ESK

ES

ESSK

Substrate Inhibition

At low substrate concentrations [S]2Ks1ltlt1 and inhibition is not observed

Plot of 1v vs 1[S] gives a line Slope = Krsquo

mVm

Intercept = 1Vm

][

111

][1

SV

K

Vv

SK

Vv

m

m

m

m

m

Substrate Inhibition

At high substrate concentrations Krsquom[S]ltlt1 and

inhibition is dominant

Plot of 1v vs [S] gives a straight line Slope = 1KS1 middot Vm

Intercept = 1Vm

mSm

S

m

VK

S

Vv

KS

Vv

1

1

][11

][1

1

max][

0][

SmKKS

Sddv

1V Igt0

I=0

1Vm

-1Km -1Kmapp 1[S]

1VIgt0

I=0

1Vm

-1Km-1Kmapp 1[S]

1Vmapp

1VIgt0

I=0

1Vm

-1Km 1[S]

1Vmapp

1V

1Vm

-1Km 1[S]

Competitive Uncompetitive

Non-Competitive Substrate Inhibition

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] onlyand competes with [S]increasing [S] overcomesInhibition by [II]

[II] binds to free [E] or [ES] complex Increasing [S] cannot overcome [II] inhibition

[II] binds to [ES] complex only increasing [S] favorsthe inhibition by [II]

E + S rarr ES rarr E + P + IIdarrEII

larr

uarr

E + S rarr ES rarr E + P + + II IIdarr darrEII + S rarrEIIS

larr

uarr uarr

E + S rarr ES rarr E + P + II darr EIIS

larr

uarr

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Kmrsquo [S] mM

vo

[S] mM

vo

II II

Km [S] mM

Vmax

II

Kmrsquo

VmaxrsquoVmaxrsquo

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax amp Km decreased

II

1[S]1Km

1vo

1 Vmax

II

Two parallellines

II

Intersect at X axis

1vo

1 Vmax

1[S]1Km 1[S]1Km

1 Vmax

1vo

Intersect at Y axis

= Kmrsquo

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

Uncompetitive Inhibitors

bull The inhibitor cannot bind to the enzyme directly but can only bind to the enzyme-substrate complex

bull ES + I = ESI

bull Both Vmax and KM are decreased by (1+IKi)

Uncompetitive Inhibition

Substrate Inhibition

Caused by high substrate concentrations

E + S ES E + PKm

rsquo k2

KS1

+

S

ES21

2

2

][][

][

][

]][[

][

]][[

Sm

m

mSi

KS

SK

SVv

ES

ESK

ES

ESSK

Substrate Inhibition

At low substrate concentrations [S]2Ks1ltlt1 and inhibition is not observed

Plot of 1v vs 1[S] gives a line Slope = Krsquo

mVm

Intercept = 1Vm

][

111

][1

SV

K

Vv

SK

Vv

m

m

m

m

m

Substrate Inhibition

At high substrate concentrations Krsquom[S]ltlt1 and

inhibition is dominant

Plot of 1v vs [S] gives a straight line Slope = 1KS1 middot Vm

Intercept = 1Vm

mSm

S

m

VK

S

Vv

KS

Vv

1

1

][11

][1

1

max][

0][

SmKKS

Sddv

1V Igt0

I=0

1Vm

-1Km -1Kmapp 1[S]

1VIgt0

I=0

1Vm

-1Km-1Kmapp 1[S]

1Vmapp

1VIgt0

I=0

1Vm

-1Km 1[S]

1Vmapp

1V

1Vm

-1Km 1[S]

Competitive Uncompetitive

Non-Competitive Substrate Inhibition

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] onlyand competes with [S]increasing [S] overcomesInhibition by [II]

[II] binds to free [E] or [ES] complex Increasing [S] cannot overcome [II] inhibition

[II] binds to [ES] complex only increasing [S] favorsthe inhibition by [II]

E + S rarr ES rarr E + P + IIdarrEII

larr

uarr

E + S rarr ES rarr E + P + + II IIdarr darrEII + S rarrEIIS

larr

uarr uarr

E + S rarr ES rarr E + P + II darr EIIS

larr

uarr

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Kmrsquo [S] mM

vo

[S] mM

vo

II II

Km [S] mM

Vmax

II

Kmrsquo

VmaxrsquoVmaxrsquo

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax amp Km decreased

II

1[S]1Km

1vo

1 Vmax

II

Two parallellines

II

Intersect at X axis

1vo

1 Vmax

1[S]1Km 1[S]1Km

1 Vmax

1vo

Intersect at Y axis

= Kmrsquo

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

Uncompetitive Inhibition

Substrate Inhibition

Caused by high substrate concentrations

E + S ES E + PKm

rsquo k2

KS1

+

S

ES21

2

2

][][

][

][

]][[

][

]][[

Sm

m

mSi

KS

SK

SVv

ES

ESK

ES

ESSK

Substrate Inhibition

At low substrate concentrations [S]2Ks1ltlt1 and inhibition is not observed

Plot of 1v vs 1[S] gives a line Slope = Krsquo

mVm

Intercept = 1Vm

][

111

][1

SV

K

Vv

SK

Vv

m

m

m

m

m

Substrate Inhibition

At high substrate concentrations Krsquom[S]ltlt1 and

inhibition is dominant

Plot of 1v vs [S] gives a straight line Slope = 1KS1 middot Vm

Intercept = 1Vm

mSm

S

m

VK

S

Vv

KS

Vv

1

1

][11

][1

1

max][

0][

SmKKS

Sddv

1V Igt0

I=0

1Vm

-1Km -1Kmapp 1[S]

1VIgt0

I=0

1Vm

-1Km-1Kmapp 1[S]

1Vmapp

1VIgt0

I=0

1Vm

-1Km 1[S]

1Vmapp

1V

1Vm

-1Km 1[S]

Competitive Uncompetitive

Non-Competitive Substrate Inhibition

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] onlyand competes with [S]increasing [S] overcomesInhibition by [II]

[II] binds to free [E] or [ES] complex Increasing [S] cannot overcome [II] inhibition

[II] binds to [ES] complex only increasing [S] favorsthe inhibition by [II]

E + S rarr ES rarr E + P + IIdarrEII

larr

uarr

E + S rarr ES rarr E + P + + II IIdarr darrEII + S rarrEIIS

larr

uarr uarr

E + S rarr ES rarr E + P + II darr EIIS

larr

uarr

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Kmrsquo [S] mM

vo

[S] mM

vo

II II

Km [S] mM

Vmax

II

Kmrsquo

VmaxrsquoVmaxrsquo

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax amp Km decreased

II

1[S]1Km

1vo

1 Vmax

II

Two parallellines

II

Intersect at X axis

1vo

1 Vmax

1[S]1Km 1[S]1Km

1 Vmax

1vo

Intersect at Y axis

= Kmrsquo

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

Substrate Inhibition

Caused by high substrate concentrations

E + S ES E + PKm

rsquo k2

KS1

+

S

ES21

2

2

][][

][

][

]][[

][

]][[

Sm

m

mSi

KS

SK

SVv

ES

ESK

ES

ESSK

Substrate Inhibition

At low substrate concentrations [S]2Ks1ltlt1 and inhibition is not observed

Plot of 1v vs 1[S] gives a line Slope = Krsquo

mVm

Intercept = 1Vm

][

111

][1

SV

K

Vv

SK

Vv

m

m

m

m

m

Substrate Inhibition

At high substrate concentrations Krsquom[S]ltlt1 and

inhibition is dominant

Plot of 1v vs [S] gives a straight line Slope = 1KS1 middot Vm

Intercept = 1Vm

mSm

S

m

VK

S

Vv

KS

Vv

1

1

][11

][1

1

max][

0][

SmKKS

Sddv

1V Igt0

I=0

1Vm

-1Km -1Kmapp 1[S]

1VIgt0

I=0

1Vm

-1Km-1Kmapp 1[S]

1Vmapp

1VIgt0

I=0

1Vm

-1Km 1[S]

1Vmapp

1V

1Vm

-1Km 1[S]

Competitive Uncompetitive

Non-Competitive Substrate Inhibition

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] onlyand competes with [S]increasing [S] overcomesInhibition by [II]

[II] binds to free [E] or [ES] complex Increasing [S] cannot overcome [II] inhibition

[II] binds to [ES] complex only increasing [S] favorsthe inhibition by [II]

E + S rarr ES rarr E + P + IIdarrEII

larr

uarr

E + S rarr ES rarr E + P + + II IIdarr darrEII + S rarrEIIS

larr

uarr uarr

E + S rarr ES rarr E + P + II darr EIIS

larr

uarr

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Kmrsquo [S] mM

vo

[S] mM

vo

II II

Km [S] mM

Vmax

II

Kmrsquo

VmaxrsquoVmaxrsquo

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax amp Km decreased

II

1[S]1Km

1vo

1 Vmax

II

Two parallellines

II

Intersect at X axis

1vo

1 Vmax

1[S]1Km 1[S]1Km

1 Vmax

1vo

Intersect at Y axis

= Kmrsquo

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

Substrate Inhibition

At low substrate concentrations [S]2Ks1ltlt1 and inhibition is not observed

Plot of 1v vs 1[S] gives a line Slope = Krsquo

mVm

Intercept = 1Vm

][

111

][1

SV

K

Vv

SK

Vv

m

m

m

m

m

Substrate Inhibition

At high substrate concentrations Krsquom[S]ltlt1 and

inhibition is dominant

Plot of 1v vs [S] gives a straight line Slope = 1KS1 middot Vm

Intercept = 1Vm

mSm

S

m

VK

S

Vv

KS

Vv

1

1

][11

][1

1

max][

0][

SmKKS

Sddv

1V Igt0

I=0

1Vm

-1Km -1Kmapp 1[S]

1VIgt0

I=0

1Vm

-1Km-1Kmapp 1[S]

1Vmapp

1VIgt0

I=0

1Vm

-1Km 1[S]

1Vmapp

1V

1Vm

-1Km 1[S]

Competitive Uncompetitive

Non-Competitive Substrate Inhibition

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] onlyand competes with [S]increasing [S] overcomesInhibition by [II]

[II] binds to free [E] or [ES] complex Increasing [S] cannot overcome [II] inhibition

[II] binds to [ES] complex only increasing [S] favorsthe inhibition by [II]

E + S rarr ES rarr E + P + IIdarrEII

larr

uarr

E + S rarr ES rarr E + P + + II IIdarr darrEII + S rarrEIIS

larr

uarr uarr

E + S rarr ES rarr E + P + II darr EIIS

larr

uarr

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Kmrsquo [S] mM

vo

[S] mM

vo

II II

Km [S] mM

Vmax

II

Kmrsquo

VmaxrsquoVmaxrsquo

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax amp Km decreased

II

1[S]1Km

1vo

1 Vmax

II

Two parallellines

II

Intersect at X axis

1vo

1 Vmax

1[S]1Km 1[S]1Km

1 Vmax

1vo

Intersect at Y axis

= Kmrsquo

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

Substrate Inhibition

At high substrate concentrations Krsquom[S]ltlt1 and

inhibition is dominant

Plot of 1v vs [S] gives a straight line Slope = 1KS1 middot Vm

Intercept = 1Vm

mSm

S

m

VK

S

Vv

KS

Vv

1

1

][11

][1

1

max][

0][

SmKKS

Sddv

1V Igt0

I=0

1Vm

-1Km -1Kmapp 1[S]

1VIgt0

I=0

1Vm

-1Km-1Kmapp 1[S]

1Vmapp

1VIgt0

I=0

1Vm

-1Km 1[S]

1Vmapp

1V

1Vm

-1Km 1[S]

Competitive Uncompetitive

Non-Competitive Substrate Inhibition

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] onlyand competes with [S]increasing [S] overcomesInhibition by [II]

[II] binds to free [E] or [ES] complex Increasing [S] cannot overcome [II] inhibition

[II] binds to [ES] complex only increasing [S] favorsthe inhibition by [II]

E + S rarr ES rarr E + P + IIdarrEII

larr

uarr

E + S rarr ES rarr E + P + + II IIdarr darrEII + S rarrEIIS

larr

uarr uarr

E + S rarr ES rarr E + P + II darr EIIS

larr

uarr

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Kmrsquo [S] mM

vo

[S] mM

vo

II II

Km [S] mM

Vmax

II

Kmrsquo

VmaxrsquoVmaxrsquo

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax amp Km decreased

II

1[S]1Km

1vo

1 Vmax

II

Two parallellines

II

Intersect at X axis

1vo

1 Vmax

1[S]1Km 1[S]1Km

1 Vmax

1vo

Intersect at Y axis

= Kmrsquo

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

1V Igt0

I=0

1Vm

-1Km -1Kmapp 1[S]

1VIgt0

I=0

1Vm

-1Km-1Kmapp 1[S]

1Vmapp

1VIgt0

I=0

1Vm

-1Km 1[S]

1Vmapp

1V

1Vm

-1Km 1[S]

Competitive Uncompetitive

Non-Competitive Substrate Inhibition

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] onlyand competes with [S]increasing [S] overcomesInhibition by [II]

[II] binds to free [E] or [ES] complex Increasing [S] cannot overcome [II] inhibition

[II] binds to [ES] complex only increasing [S] favorsthe inhibition by [II]

E + S rarr ES rarr E + P + IIdarrEII

larr

uarr

E + S rarr ES rarr E + P + + II IIdarr darrEII + S rarrEIIS

larr

uarr uarr

E + S rarr ES rarr E + P + II darr EIIS

larr

uarr

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Kmrsquo [S] mM

vo

[S] mM

vo

II II

Km [S] mM

Vmax

II

Kmrsquo

VmaxrsquoVmaxrsquo

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax amp Km decreased

II

1[S]1Km

1vo

1 Vmax

II

Two parallellines

II

Intersect at X axis

1vo

1 Vmax

1[S]1Km 1[S]1Km

1 Vmax

1vo

Intersect at Y axis

= Kmrsquo

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d D

escr

iptio

n

[II] binds to free [E] onlyand competes with [S]increasing [S] overcomesInhibition by [II]

[II] binds to free [E] or [ES] complex Increasing [S] cannot overcome [II] inhibition

[II] binds to [ES] complex only increasing [S] favorsthe inhibition by [II]

E + S rarr ES rarr E + P + IIdarrEII

larr

uarr

E + S rarr ES rarr E + P + + II IIdarr darrEII + S rarrEIIS

larr

uarr uarr

E + S rarr ES rarr E + P + II darr EIIS

larr

uarr

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Kmrsquo [S] mM

vo

[S] mM

vo

II II

Km [S] mM

Vmax

II

Kmrsquo

VmaxrsquoVmaxrsquo

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax amp Km decreased

II

1[S]1Km

1vo

1 Vmax

II

Two parallellines

II

Intersect at X axis

1vo

1 Vmax

1[S]1Km 1[S]1Km

1 Vmax

1vo

Intersect at Y axis

= Kmrsquo

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Kmrsquo [S] mM

vo

[S] mM

vo

II II

Km [S] mM

Vmax

II

Kmrsquo

VmaxrsquoVmaxrsquo

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax amp Km decreased

II

1[S]1Km

1vo

1 Vmax

II

Two parallellines

II

Intersect at X axis

1vo

1 Vmax

1[S]1Km 1[S]1Km

1 Vmax

1vo

Intersect at Y axis

= Kmrsquo

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

Factors Affecting Enzyme

Kinetics

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

Effects of pH

- on enzymes

- enzymes have ionic groups on their active sites

- Variation of pH changes the ionic form of the active sites

- pH changes the three-Dimensional structure of enzymes

- on substrate

- some substrates contain ionic groups

- pH affects the ionic form of substrate

affects the affinity of the substrate to the enzyme

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

Effects of Temperature

Reaction rate increases with temperature up to a limit

Above a certain temperature activity decreases with temperature

due to denaturation

Denaturation is much faster than activation

Rate varies according to the Arrhenius equation

tkRTE

RTEdd

tk

RTE

da

a

d

a

eEAev

eAk

eEE

Aek

Ekv

0

0

2

2

][][

][Where Ea is the activation energy (kcalmol)

[E] is active enzyme concentration

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

Factors Affecting Enzyme Kinetics Temperature

- on the rate of enzyme catalyzed reaction

k2=Aexp(-EaRT)

T k2

- enzyme denaturation

T

][][

2ESk

dt

Pdv

v

][][

Edkdt

Ed

Denaturation rate

kd=Adexp(-EaRT)

kd enzyme denaturation rate constant

Ea deactivation energy

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

REFERENCES

Michael L Shuler and Fikret Kargı Bioprocess Engineering Basic Concepts (2 nd Edition)Prentice Hall New York 2002

1 James E Bailey and David F Ollis Biochemical Engineering Fundementals (2 nd Edition) McGraw-Hill New York 1986

wwwbiochemumasseducourses420lecturesCh08Bppt -

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

classfstohio-stateedufst605605p

Enzymespdf ndash

wwwhortonednetnscastaffselig

powerpointsbio12biochemenzymespdf

wwwsiuedudepartmentsbiochemsom_pbl

SSBpowerpointenzymesppt

wwwassociazioneasiaitadonfiles

2005_luisi_05_why_are_enzymespdf

wwwfatihedutr~abasiyanik

Chapter6_enzymespdf -

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml

httpwwwauthorstreamcompresentationkkozar-14001-enzymes-enzyme-ppt-education-powerpoint

httphigheredmcgraw-hillcomsites0072495855student_view0chapter2animation__how_enzymes_workhtml

httpwwwwileycomcollegepratt0471393878

studentanimationsenzyme_kineticsindexhtml