principles of biochemistry (semester 2 -2014/15) · principles of biochemistry (semester 2...
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BCH 3000PRINCIPLES OF BIOCHEMISTRY
(Semester 2 -2014/15)
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Enzymology -1
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Enzymology -2
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Catalysis and Enzymology
• Introduction to catalysis
– Free energy profiles
– What enzymes do
• Chemical kinetics and reaction order
– First-order kinetics
– Other reaction orders
• Enzyme kinetics
– Binding and saturation
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What is a catalyst?
• Example:
Features of a catalyst:
• Makes an alternative reaction path in which less
activation energy (DG‡) is needed
• Equally increases the rate of the back and forth
reaction reaction catalysis has no effect on
equilibrium position!
H3C C
O
OH + C2H5OH H3C C
O
OC2H5 + H2OH+
“A catalyst is a compound which enhances the rate of a
chemical reaction without being destroyed or incorporated in
the product” (IUPAC)
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DG‡
Gibbs
free
energy
S
P
Reaction coordinate
X‡ (T.S.)
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Two ways to decrease DG‡:
a By lowering the energy content
of the transition state
Eact
Eact
E
progress of reaction
a b
b By ground state
destabilisation15
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What kinds of catalysts are there
around?• Organic
• Inorganic
• Biological (biocatalyst)
Other way of subdivision:
• Homogeneous = freely dissolved in solution
– organic catalyst
– organometallic complex
– enzyme in water
• Heterogeneous = solid, in liquid or gaseous
environment
– inorganic catalyst (e.g. zeolite)
– immobilised enzyme
– enzyme in an organic solvent16
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All Enzymes are not Proteins:
Ribozymes
• Ribozymes defined as RNA catalysts
• Function as phosphodiester
transferases
– Sequence specific
– Kinetically like enzymes
– Efficiently lower DG*
– Require native 3D-structure
• Function in RNA splicing
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How Enzymes Work
• Enzymes are protein catalysts: they
increase the velocity of the reaction, but
are not themselves altered
• Enzymes cannot change Keq/DGo
• Enzymes do change DG*/k
– Energy of transition state decreased or
– Energy of ground state increased
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Simple Kinetics: A First-order
Reaction
A B
v = -d[A]/dt = d[B]/dt units: M/time
v = k[A] Empirical rate law
(M x time-1) = k(M) units of k: time-1
Reaction order: exponent of concentration
term on which rate depends. Above rxn is 1st
order in reactant A 19
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1st-Order Rate Equation
v = -d[A]/dt = k[A]
or d[A]/[A] = d ln[A] = -kdt
so, ln [A] = ln[Ao] –kt and [A] = [Ao]e-kt
Slope = -k[A]=[Ao]e-kt;
exponential
decay
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Other Reaction Orders
• Zero order reaction independent of [A]
– v = k, units of k: M x time -1
• Second order reaction: A + B P
– v = k[A][B]
– k: unit of M-1time-1
– 1st order in A
– 1st order in B; second order overall
– For 2A P; v = k[A]2; 2nd order in A
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Order in Enzyme Catalysis
depends on [S]
S P
vo = initial velocity; measure at
several [S] but constant [E]
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Michaelis-Menten Kinetics. I.
At infinite [S], vo = Vmax
Km defined as [S] at which vo = ½ Vmax
M-M Equation:
vo = (Vmax [S])/(Km + [S])
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Michaelis-Menten Kinetics. II
if [S] << Kmvo = (Vmax/Km)[S]
if [S] = Km vo = 0.5 Vmax
if [S] >> Km vo = Vmax
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-10
-5
0
5
10
15
20
-50 -30 -10 10 30 50
v, µ
mo
l/m
in
[S], mM
Enzymes
ES ES P v Vmax[S]
Km [S]
k1
k-1
k2
-Km, Vmax
0
1
2
3
4
5
0 10 20 30 40 50v
, µ
mo
l/m
in
[S], mM
Km
0.5Vmax
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Enzymology -3
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Amino Acids, Proteins, and
Enzymes
Enzymes
Enzyme Action
Factors Affecting Enzyme Action
Enzyme Inhibition
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Enzymes
• Catalysts for biological reactions
• Most are proteins
• Lower the activation energy
• Increase the rate of reaction
• Activity lost if denatured
• May be simple proteins
• May contain cofactors such as metal ions
or organic (vitamins)
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Name of Enzymes
• End in –ase
• Identifies a reacting substance
sucrase – reacts sucrose
lipase - reacts lipid
• Describes function of enzyme
oxidase – catalyzes oxidation
hydrolase – catalyzes hydrolysis
• Common names of digestion enzymes still use –in
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Classification of Enzymes
Class Reactions catalyzed
• Oxidoreductoases oxidation-reduction
• Transferases transfer group of atoms
• Hydrolases hydrolysis
• Lyases add/remove atoms
to/from a double bond
• Isomerases rearrange atoms
• Ligases combine molecules
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Examples of Classification of
Enzymes
• Oxidoreductoases
oxidases - oxidize ,reductases – reduce
• Transferases
transaminases – transfer amino groups
kinases – transfer phosphate groups
• Hydrolases
proteases - hydrolyze peptide bonds
lipases – hydrolyze lipid ester bonds
• Lyases
carboxylases – add CO2
hydrolases – add H2O 37
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Enzyme Action:
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
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Lock and Key Model
+ +
E + S ES complex E + P
S
P
P
S
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Enzyme Action:
Induced Fit Model
• Enzyme structure flexible, not rigid
• Enzyme and active site adjust shape to
bind substrate
• Increases range of substrate specificity
• Shape changes also improve catalysis
during reaction
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Enzyme Action:
Induced Fit Model
E + S ES complex E + P
S
P
P
SS
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Factors Affecting Enzyme
Action: Temperature
• Little activity at low temperature
• Rate increases with temperature
• Most active at optimum temperatures
(usually 37°C in humans)
• Activity lost with denaturation at high
temperatures
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Factors Affecting Enzyme
Action
Optimum temperature
Reaction
Rate
Low High
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Factors Affecting Enzyme
Action: Substrate Concentration
• Increasing substrate concentration
increases the rate of reaction (enzyme
concentration is constant)
• Maximum activity reached when all of
enzyme combines with substrate
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Factors Affecting Enzyme
Action
Maximum activity
Reaction
Rate
substrate concentration
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Factors Affecting Enzyme
Action: pH
• Maximum activity at optimum pH
• R groups of amino acids have proper
charge
• Tertiary structure of enzyme is correct
• Narrow range of activity
• Most lose activity in low or high pH
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Factors Affecting Enzyme
Action
Reaction
Rate
Optimum pH
3 5 7 9 11
pH
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Enzyme Inhibition
Inhibitors
• cause a loss of catalytic activity
• Change the protein structure of an enzyme
• May be competitive or noncompetitive
• Some effects are irreversible
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Competitive Inhibition
A competitive inhibitor
• Has a structure similar to
substrate
• Occupies active site
• Competes with substrate for
active site
• Has effect reversed by increasing
substrate concentration
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Noncompetitive Inhibition
A noncompetitive inhibitor
• Does not have a structure like substrate
• Binds to the enzyme but not active site
• Changes the shape of enzyme and active
site
• Substrate cannot fit altered active site
• No reaction occurs
• Effect is not reversed by adding substrate
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Enzymology -4
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• The Michaelis-Menten Equation
• Louis Michaelis and Maude Menten's theory
• It assumes the formation of an enzyme-substrate complex
• It assumes that the ES complex is in rapid equilibrium with free enzyme
• Breakdown of ES to form products is assumed to be slower than 1) formation of ES and 2) breakdown of ES to re-form E and S
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• Enzymes are regulated:
• at genetic level (transcription, translation);
• by concentration of substrate and product;
• allosterically.
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Enzymology -5Co-enzymes &
Cofactors
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Cofactors are exogenous molecules that
associate with proteins to yield full activity. In
the absence of cofactor, protein is an
apoprotein.
Cofactors
Prosthetic groups are covalently attached to the
protein. Examples are heme, in hemoglobin, and
riboflavin, in flavoproteins.
Co-enzymes are soluble and associate transiently
with enzyme during catalytic cycle. An example is
vitamin K in activation of blood clotting enzymes.
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Some Cofactors
OP
OHO
HO
N
O
OH
H
N N
S OP
HO O
O POH
OH
O
NH2
N S
HN
NH
O
OH
O+
Thiamin Diphosphate Pyridoxal PhophatesBiotin
N
OH
OHN
OH
NO
O
HN
O
PO
O
O-
PO
O
O-
O
HO
N
OH
N
N
NH2N
Na+Na+
N
NN
N
OHO
O
OH
Fe
FAD
Heme
HO
ON
OH
P
O
OH
O P
O
OH
OHO
OO
N
N N
N
NH2
O
R
N
OH
OHN
OH
NO
O
HN
O
PO-
O
O-
FMN
R= H NADHR= -PO3H2 NADPH
O
NH2
N
N
N
NH2
N
O
OP
OH
O
O
OPO
OH
OH
O
HN
HN
O
SH
OHO
OH
PHO
O
Coenzyme A (CoA)
NN
N N
H2N
O NH2
O
NH2
NH2O
O
OCo
O
H
H2N
O
NH
O
C
PO
O
-OO
N
N
HO H
H
NH2
H
H
OH
N
Vitamin B12
O
NH
N
N
N
NH2
NH
NH
O
O
HO
O
OH
SS
H
OH
O
Folic Acid
Lipoic Acid
HN
OHS
O
NH
NH2
HO
O OH
O
Glutathione
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Thiamin Dependent Reactions
Acetolactate synthase
CH3C
O
CO2H2thiamin
CH3C
O
C
OH
CH3
CO2H + CO2
Pyruvate dehydrogenase: pyruvate decarboxylase,
dihydrolipoyl transacetylase, dihydrolipoyl dehydrogenase,
CH3C
O
CO2H
pyruvate
+ thiamin + lipoic acid + CoA-SH Acetyl-CoA + CO2(CH3COS-CoA)
N N
S OP
-O O
O PO
-
O-
O
NH2
N
+
Thiamin Diphosphate (pyrophosphate)
SH
NH
OSH3C
O
Lys
S
NH
OLys
S
CO2-
O
- CO2
thiamin, CoA-SH
S-CoA
O
Pyruvate Decarboxylase
CH3C
O
CO2Hthiamin
CH3C
O
H + CO2
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Pyridoxal Phosphate (PLP) dependent enzymes (Vitamin B6)
(Bugg, Chapt. 9, pp 186-198)
Involved in amino acid biosynthesis, metabolism and catabolism
N
2-O3POOH
O H
N
2-O3POOH
NH2
pyridoxal
N
2-O3POOH
OH
pyridoxamine pyridoxine
R
NH3
CO2-
H
L-amino acid
R
NH3
HCO2
-
D-amino acid
racemase,epimerase
R
NH3
HH
decarboxylase
R
O
CO2-
transaminase
R
NH2
CO2-
H2O
R'
NH3
CO2-
H
reaction at the or carbon
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