azin nowrouzi, phd department of biochemistry tehran

TUMS

Azin Nowrouzi, PhD Department of Biochemistry

Tehran University of Medical Sciences

Topics

Activity of enzyme and its expression

Unit

Katal

Specific activity

Factors that affect activity

Enzyme Kinetics

definition MM equation

Lineweaver-Burk equation

Enzyme inhibition

Reversible

Competitive

Mixed or non-competitive

Uncompetitive

Irreversible

Allosteric enzymes

Multi-substrate reactions

Ping-pong mechanism

Sequential

Random

Ordered

Enzyme activity

1. Enzyme Unit:

A. One unit = 1 micromol/min= 1µmol/min • the amount of enzyme that converts 1 µmol substrate to product in 1 minute.

B. One unit = 1 nanomol/min= 1nmol/min

2. 1 katal = 1 mol/s

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A measure of the ability of a given enzyme to convert its substrate(s) into its product(s).

To find the activity of an enzyme: We measure either the amount of substrate that's disappearing, or the amount of product that's appearing over a specified period of time. Activity is expressed as Units or Katal.

Enzyme activity is a measure of the quantity of active enzyme present.

Activity is the same as: • speed • velocity • Rate

of the reaction

Enzyme specific activity

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Example for enzyme specific activity:

In a sample of blood the protein concentration is 4 g/dl, and the enzyme activity is 60 U/ml. what is the specific activity of the enzyme? a. 1.5 IU/ml b. 15 IU/ml c. 150 IU/ml d. 1500 IU/ml

Specific activity is the number of enzyme units per ml divided by the concentration

of protein in mg/ml. Specific activity values are therefore quoted as units/mg.

Factors that influence activity

Important factors in reaction rates:

1. Temperature

2. pH

3. Enzyme concentration [E]

4. Substrate concentration [S]

5. Inhibitors

6. Regulation (Effectors)

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A typical graph of rate against temperature

• The temperature at which the rate is fastest is called the optimum temperature for that enzyme.

1

2

3

① Little activity at low temperature (low number of collisions)

② Rate increases with temperature (more successful collisions) rate doubles or

triples for every 10°C increase in temperature

③ Activity lost with denaturation at high temperatures

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Optimum temperature

Enzymes are most active at optimum temperatures (usually 37 C in humans) Enzymes isolated from thermophilic organisms display maxima around 100°C Enzymes isolated from psychrophilic (cryophilic) organisms display maxima

around 10°C

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Optimum pH

• Effect of pH on ionization of active site.

• Effect of pH on enzyme denaturation.

• Each enzyme has an optimal pH (most of the times ≈ 6 - 8 ) – Exceptions :

digestive enzymes in the stomach( pH 2)

digestive enzymes intestine (pH 8)

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Effect of pH on ionization of active site

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Too much Basic condition

Too much Acidic condition

Correct pH in the microenvironment

Enzyme concentration

• The Rate (v) of reaction Increases proportional to the enzyme concentration [E] ([S] is high).

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Enzyme saturation with substrate

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Enzyme kinetics

• Kinetics:

The study of reaction rates (velocities)

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Dependence of velocity on [substrate] concentration.

many enzymes follow Michaelis-Menten equation:

2 1 3 4 5 6 7 8 0

0 2 4 6 8

Substrate (mmole) [S]

Pro

du

ct (

v)

80

60

40

20

0

S + E

(in a fixed

period of

time)

Enzyme Velocity Curve

P

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Michaelis-Menten equation

S E k1

k-1 E S

k2 P

0

1

2

3

4

5

0 10 20 30 40 50

v,

µm

ol/

min

[S], mM

0.5Vmax

Km

Vmax ,سرعت ماکزیمم

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منتن-منحنی سهمی میکائیلیس

Order of reaction

1. When [S] << Km

vo = (Vmax / Km )[S]

2. When [S] = Km

vo = Vmax /2

3. When [S] >> Km

vo = Vmax

zero order

First order

Mixed order

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MM equation derivation (steady state)

Substrate concentration

• When enzyme concentration is constant, increasing [S] increases the rate of reaction, BUT

• Maximum activity reaches when all E combines with S (when all the enzyme is in the ES, ,form).

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Example

• In an enzyme catalyzed reaction [S] is 1mmol and initial velocity is equal to 2/3Vmax. Calculate Km?

• In an enzymatic reaction if substrate concentration is equal to Km, what would be the relationship between Vi and Vmax?

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Lineweaver-Burk plot

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1

vKm

Vmax

1

[S]1

Vmaxv

Vmax [S]

Km [S]

Direct plot Double reciprocal

Enzymes are subject to reversible inhibition

Competitive inhibition

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• Competitive Inhibition:

o A competitive inhibitor binds to the active site of the enzyme.

o A competitive inhibitor does not change the maximum rate for the reaction.

o A competitive inhibitor lowers the enzymes affinity for its substrate.

Competitive inhibition of succinate dehydrogenase

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Example: Competitive inhibition

• The antibiotic sulfanilamide is similar in structure to para-aminobenzoic acid (PABA), an intermediate in the biosynthetic pathway for folic acid.

• Sulfanilamide can competitively inhibit the enzyme that has PABA as its normal substrate by competitively occupying the active site of the enzyme.

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Example: Enzyme inhibitors in medicine

•Many current pharmaceuticals are enzyme inhibitors

(e.g. HIV protease inhibitors for treatment of AIDS)

•An example: Ethanol is used as a competitive

inhibitor to treat methanol poisoning.

Methanol formaldehyde (very toxic)

Ethanol competes for the same enzyme.

Administration of ethanol occupies the enzyme

thereby delaying methanol metabolism long enough

for clearance through the kidneys.

Alcohol dehydrogenase

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Uncompetitive inhibition

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• Un-Competitive or Anti-competitive Inhibition:

o binds to a site other than the active site.

o slows the maximum attainable rate of the reaction.

o lowers the enzymes affinity for its substrate.

Mixed inhibition

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o A non-competitive inhibitor slightly decreases an enzymes affinity for its substrate by altering the shape of the active site.

Pure Non-competitive inhibition

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• Non-Competitive Inhibition:

o A non-competitive inhibitor binds to a site other than the active site.

o A non-competitive inhibitor slows the maximum attainable rate of the reaction.

Reversible Inhibition = Temporary decrease of enzyme function

• Three types based on “how increasing [S] affects degree of inhibition”:

1. Competitive – degree of inhibition decreases

2. Mixed or Non-competitive – degree of inhibition is unaffected

3. Anti- or Uncompetitive – degree of inhibition increases

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Types of enzyme inhibitors

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Uncompetitive

Irreversible Inhibition = Enzyme stops working permanently

1. Destruction of enzyme (heat or extremes of pH) 2. Irreversible Inhibitor = forms covalent bonds to E (inactive E) Examples:

– Diisopropylfluorophosphate • inhibits acetylcholine esterase • binds irreversibly to –OH of serine residue

– Cyanide and sulfide • Inhibit cytochrome oxidase • bind to the iron atom

– Fluorouracil • inhibits thymidine synthase (suicide inhibition - metabolic product

is toxic )

– Aspirin • Inhibits prostaglandin synthase • acylates an amino group of the cyclooxygenase

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(1) Irreversible Inhibition: inhibitor binds tightly, often covalently, to the enzyme,

permanently inactivating it.

DIPF = DIFP = diisopropylfluorophosphate

kinetics

• Dependence of velocity on [substrate] is described for many enzymes by the Michaelis-Menten equation:

• kinetic parameters: – Km (the Michaelis constant) – Kcat (the turnover number, which relates Vmax, the maximum velocity, to [Et],

the total active site concentration) – kcat/Km (the catalytic efficiency of the enzyme) – can't be greater than limit imposed by diffusion control, ~108-109 M–1sec–1

• Kinetic parameters can be determined graphically by measuring velocity of enzyme-catalyzed reaction a(V vs. [substrate]).

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Why study kinetics

• Study of enzyme kinetics is useful for measuring 1. concentration of an enzyme in a mixture (by its catalytic activity), 2. its purity (specific activity), 3. its catalytic efficiency and/or specificity for different substrates 4. comparison of different forms of the same enzyme in different tissues or

organisms, 5. effects of inhibitors (which can give information about catalytic mechanism,

structure of active site, potential therapeutic agents...)

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Allosteric enzymes

• Why the sigmoid shape?

• Allosteric enzymes are multi-subunit enzymes, each with an active site.

• They show a cooperative response to substrates

Sigmoidal curve

hyperbolic curve michaelis-menten

kinetics

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Allosteric enzymes

• They have oligomeric organization – They have more than one site, where effector binding at one site

induces a conformational change in the enzyme, altering its affinity for a substrate.

• Their kinetics do not obey the Mikaelis-Menten equation • Their v versus [S] plot yields sigmoid or S-shaped curves rather than

rectangular hyperbolic – sensitive to a small change in [S].

• Substrate binding is cooperative – Binding of one substrate alters the enzyme’s conformation and

enhances the binding of subsequent substrates.

• Inhibition of a regulatory enzyme does not conform to any normal

inhibition pattern. – Allosteric inhibition – Allosteric activation

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CO2 Also Promotes the Dissociation of O2 from Hemoglobin

Oxygen binding curves of blood and of hemoglobin in the absence and presence of CO2 and BPG.

Multi-substrate reactions

• Ping Pong mechanism for two substrates

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Multi-substrate reactions

• Sequential mechanism

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Regulation of enzyme activity

Enzyme concentration

Gene trascription

Gene induction Gene

suppression

Control of translation

(mRNA stability)

Protein half life

Enzyme activity

Compartmentalization

Isoenzymes

Lactate dehydrogease

Creatine phosphate

Hexokinase Glucokinase

Allosteric regulation)

Product inhibition

Endproduct inhibition (feedback inhibition)

Substrate

(feedforward activation)

Covalent modification

Regulatory subunit

proteolysis

Allosteric regulation

• Allosteric enzymes – Enzymes whose activity can be changed by molecules (effector molecules) other than substrate.

• Allosteic means “other site” or “other structure”

• The interaction of an inhibitor at an allosteric site changes the structure of the enzyme so that the active site is also changed.

• Negative allosterism

• Positive allosterism

• Binding of the effector molecule regulates enzyme activity by determining whether it will be active or not.

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Enz 2 Enz 1 Enz 3 Enz 4 B C D E A

Product inhibition and end product inhibition

-

Negative feedback inhibition

-

Product inhibition

Rate determining steps

Rate limiting steps

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Product inhibition

Hexokinase- first reaction in glycolysis, hexokinaseis inhibited by glucose-6-phosphate (G6P, the product)

ADP ATP Glucose Glucose-6-phosphate

Hexokinase

×

Product inhibition

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اولین مرحله در سنتس اوراسیل و

سیتوزین

Example

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End-product inhibition

Proenzymes (zymogen)

Proteolytic activation Activation of a zymogen.

• Some enzymes are secreted as inactive precursors, called zymogens. • Pancreatic proteases - trypsin, chymotrypsin, elastase,

carboxypeptidase are all synthesized as zymogens - trypsinogen, chymotrypsinogen, proelastase and procarboypeptidase

• Clotting factors Fibrinogen and pro-thrombin • Hormone peptides (Conversion of Pro-insulin to

Insulin) • an on/off switch more than regulation. • Fibrous proteins: Collagen is synthesized as

procollagen

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Real life examples of activation by proteolytic cleavage

Trypsin activation

45 Chymotrypsin activation

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Different types of covalent modification

Phosphorylation

Conformational Change dephosphorylastion

Phosphatase

P

Protein

OH

SerThr Tyr(His)

Inactive Active

Glycogen phosphorylase b Glycogen phosphorylase a

Juang RH (2004) BCbasics

Kinase

phosphorylation

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ADP ATP Glucose Glucose-6-phosphate

Hexokinase

Isoenzymes (isozymes)

• Multiple forms of same enzyme • Catalyse the same chemical reaction • Different chemical and physical properties:

– Electrophoretic mobility – Kinetic properties – Amino acid sequence – Amino acid composition

Hexokinase Glucokinase

Location in cell

All cell types (cytoplasm)

Liver (mitochondria)

Km 5 x 10-5M 0.05 mM

5 x 10-3M 5mM

Glucokinase

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Aminotransferases

Aspartate aminotransferase

(AST or SGOT)

Alanine aminotransferase

(ALT, or SGPT)

Myocardial infarction

Viral hepatitis

Lactate Dehydrogenase (LDH) myocardial infarction

Creatine Kinase (CK) Myocardial infarc., brain,

skeletal muscle disorder

Cholinesterase Liver, erythrocytes

Gamma-glutamyltransferase Liver damage

Acid phosphatase Carcinoma of prostate

Alkaline phosphatase (AP) Bone disease

Lipase Acute pancreatitis

Alpha-amylase Intestinal obstruction Som

e d

iag

no

stically

im

port

an

t e

nzym

es

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Isozymes of lactate dehydrogenase

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In LDH • Subunits M and H can combine to make five different tetramers.

H4

M4

H3M

HM3

(LDH, EC 1.1.1.27.)

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• CK has 3 forms dimer B and M chains: • CK1= BB • CK2= MB • CK3=MM • Heart is the only tissue rich in CK2, increases 4-8 hr

after chest pains- peaks at 24 hr. • LDH peaks 2-3 days after MI.

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Isoenzymes of Creatine kinase

Electrophoresis of LDH and CK isoenzymes in blood

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Isoenzyme Name Circulating Lifetime

Mitochondrial AST 1hr

LDH-5 (M4) 12 hrs

CK 18 hrs

Cytoplasmic AST 1 day

ALT 2 days

LDH-1 (H4) 5 days

Increase in important isoenzymes in blood after chest pain

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