Enzymes(Inhibition, regulation, isoenzymes &

specificity)

DrArun Kumar K

Enzyme Inhibition

Enzyme activity is decreased by certain compounds known as inhibitors. The process is called enzyme inhibition.

1.Competitive Inhibition

2.Non competitive inhibition

3.Uncompetitive inhibition

4.Suicide inhibition

5.Allosteric inhibition

1. Competitive inhibition

• Competition b/w substrate and inhibitor for substrate binding site.

• Inhibitor is a structural analogue of substrate.

• E+I EI ; No product

• Reversible. Can be reversed by increasing substrate conc.

• Increased Km, No change in V max.

• Example :

Succinate dehydrogenase inhibited by Malonate

Examples for Clinical Applications of Competitive Inhibition (drugs)

• 1)Allopurinol – Xanthine Oxidase (Gout).

• 2) Dicumarol – Vitamin K Epoxide reductase(Anticoagulant)

• 3) Sulfonamide- Pteroid synthase (Antibiotic)

• 4) Methotrexate & Aminopterin- Dihydrofolatereductase (Anticancer drug)

• 5) Neostigmine –Acetyl cholinesterase (Myasthenia Gravis)

• 6) Lovastatin – HMG CoA Reductase, (Hypercholesterolemia)

• 7) Succinyl choline: Acetyl choline esterase (muscle relaxation in surgery)

• 8) Ephidrene, Amphetamine: Mono Amino Oxidase (Hypotension)

• 9) Ethanol in methanol poisoning: Alcohol Dehydrogenase.

Noncompetitive Inhibition

• There is no competition.

• Inhibitor is not a structural analogue.

• binds to site other than active site.

• Generally Irreversible. Can not be reversed by increasing substrate concentration.

• V Max decreased ; Km unchanged.

Examles for noncompetitive inhibition (poisons)

Cyanide – Cytochrome oxidase

Diisopropyl fluorophosphate (DIFP) -Acetylcholinesterase.

Fluoride- Enolase

Iodoacetate : -SH enzymes (Glyceraldehdye 3-phosphate Dehydrogenase).

3. Uncompetitive Inhibition

• Inhibitor Binds only to ES (not to free enzyme).

• Both Km and V Max decreased.

• Example : Inhibition of placental alkaline phosphatase by Phenylalanine.

Suicide Inhibition (Mechanism based inactivation)

• Structural analog of the substrate is converted to a more effective inhibitor by the action of the enzyme to be inhibited.

• This new inhibitor formed irreversibly binds to the enzyme and inhibits it.

• Irreversible inhibition.

• Examples:

• 1) Allopurinol (→Alloxanthine) – Xanthineoxidase

Hypoxanthine ---→ xanthine

• 2) Aspirin (anti inflammatory agent) -cyclooxygenase.

Arachidonic acid ----→ prostaglandins.

• 3) Di Fluoro Methyl Ornithine (DFMO-treatment of trypanosomiasis) –OrnithineDecarboxylase

Ornithine -----→ Putrescine

5. Allosteric inhibition

• Type of enzyme regulation.

Enzyme regulation

• Activity of certain can be increased or decreased depending on the need of the cell.

• These enzymes are called Regulatory enzymes.

• Types:

• 1. Allosteric regulation

• 2. Covalent modification

• 3. Induction

• 4. Repression.

SUBSTRATE BINDING SITE ALLOSTERIC

SITE

ALLOSTERIC ENZYME

Allosteric Regulation (Allos = other)

1. Allosteric Regulation (Allos = other)

• Allosteric enzyme has a site other than catalytic site known as allosteric site.

• It is for modifiers to bind.

• The binding of the regulatory molecule can either

• enhance the activity of the enzyme –allosteric activation, or

• inhibit the activity of the enzyme - allostericinhibition.

Allosteric activation

• The binding of the regulatory molecule to allosteric site enhances activity of the enzyme.

• The modifier is called- positive modifier.

ALLOSTERIC ENZYME ALLOSTERIC ACTIVATOR

Phosphofructokinase AMP & Fructose 2,6 Bis hosphate

Aspartate transcarbamoylase ATP

Pyruvate carboxylase Acetyl CoA

Acetyl CoA-carboxylase Citrate

Carbamoyl phosphatesynthetase I

N- Acetyl Glutamate

Allosteric inhibition

• The binding of regulatory molecule to allosteric site decreases activity of the enzyme.

• The modifier is negative modifier.

• When an inhibitor binds to allosteric site,

• configuration of catalytic site is modified

• such that substrate cannot bind properly.

• The inhibitor is not a substrate analog.

• It is partially reversible, when excess substrate is added.

• Km is usually increased, Vmax is reduced

ALLOSTERIC ENZYME ALLOSTERIC INHIBITOR

Phosphofructokinase ATP & Citrate

Aspartate transcarbamoylase CTP

Pyruvate carboxylase ADP

Acetyl CoA-carboxylase Acyl CoA

Carbamoyl phosphatesynthetase II

UTP

feedback inhibition or end-product inhibition –activity of the enzyme is inhibited by the final product of the biosynthetic pathway.

2. Covalent modification

• It is a type of enzyme regulation.

• There is either addition of a group to the enzyme by forming a covalent bond; or

• removal of a group by cleaving a covalent bond.

• Types:

i. Irreversible

ii. Reversible

i. Irreversible covalent modification

• Activation of enzyme by breaking a covalent bond present the enzyme.

• Once activated cannot be converted back to original inactive form.

• Examples

• Zymogen activation by partial proteolysis.

PROENZYMES / ZYMOGENS

ACTIVE ENZYMES

1 PEPSINOGEN PEPSIN

2 TRYPSINOGEN TRYPSIN

3 CHYMOTRYPSINOGEN CHYMOTRYPSIN

4 PROELASTASE ELASTASE

PROENZYME ACTIVE ENZYME +Partial Proteolysis

POLYPEPTIDEPeptide bond

ii. Reversible covalent modification

• By Addition or removal of a group such as

i. phosphate group (phosphorylation–dephosphorylation) – (most common).

ii. ADP-ribose group (rare)

ATP

ADPH20

Pi

PROTEIN KINASEPROTEIN PHOSPHATASE

PhosphorylationDephosphorylation

Enzyme Phosphorylatedform

Acetyl-CoAcarboxylase

Inactive

Glycogen synthase Inactive

Pyruvatedehydrogenase

Inactive

HMG CoAreductase

Inactive

Pyruvate kinase Inactive

Phosphorylated form is Inactive

Phosphorylated form is Active

Enzyme Phosphorylatedform

Glycogen phosphorylase

Active

Phosphorylase b kinase

Active

HMG CoAreductase kinase

Active

3. Induction

• Process of increasing rate of synthesis of enzyme in the presence of inducer.

• Induction is effected through the process of derepression.

• The inducer will relieve the repression on the operator site and

• will remove the block on the biosynthesis of the enzyme molecules.

Inducible enzyme

inducer

1 Tryptophan pyrrolase

glucocorticoids

2 transaminase glucocorticoids

3 Glucokinase insulin

4 ALAsynthase

barbiturates

4. Repression

• Process of decreasing rate of synthesis of enzyme in the presence of repressor.

• Examples:

• ALA synthase is repressed by heme.

• HMG CoA reductase is repressed by Cholesterol.

• Inhibition by the end product of the pathway is called FEEDBACK Inhibition

Isoenzymes

• Multiple (physically distinct) forms of the same enzyme which catalyze the same reaction but differ in

• Physical

• Chemical

• Immunoloical properties and

• Electrophoretic mobilities.

Types of isoenzymes

1. Product of different genes (true isoenzymes)

2. Which differ in subunit composition.

3. Which differ in prosthetic group composition (iso-forms).

1. Product of different genes

Example:

Salivary amylase and Pancreatic amylase

2. Which differ in subunit composition

Example:

i. Creatine kinase

ii. Lactate Dehydrogenase(LDH).

3. Isoenzymes which differ inprosthetic group composition.

• Example:

• Alkaline Phosphatase (ALP)

• ALP hydrolyzes compounds containing phosphate under alkaline condition (optimum pH is between 9 and 10).

• Zinc is a constituent ion of ALP

• Contains sialic acid as prosthetic group.

Separation of isoenzymes

1. Agar gel or polyacrylamide gel electrophoresis.

2. Difference in Heat stability.

3. Using Inhibitors.

4. Difference in affinity for substrate

5. Using Specific antibodies

Enzyme specificity

• Enzymes are specific in their action.

• 4 types:

1. Absolute specificity

2. Group specificity

3. Bond specificity

4. Stereospecificity

1. Absolute specificity

• Enzymes which show absolute specificity are specific about

- (a) their substrate and

- (b) type of reaction.

Example:

1. Urease catalyzes hydrolysis of Urea.

2. Glucose oxidase catalyzes oxidation of glucose.

2. Group specificity

• These enzymes are specific about a particular group present in their substrate.

• Example:

1. Hexokinase can add phosphate to

- glucose,

- galactose and

- mannose.

3. Bond specificity

• Are specific about a particular bond present in their substrate.

• Example:

1. Trypsin hydrolyzes peptide bonds of any protein

- hydrolyzes internal peptide bonds involving carboxyl groups of arginine or lysine residues.

2. Chymotrypsin – internal peptide bond - Pheand Tyr

4. Stereospecificity

• These enzymes are specific about stereo isomeric form of their substrate.

• Example:

1. Hexokinase acts only on D- Glucose

2. Transaminases act only on L- Amino acids.

The activity of an enzyme is also sensitive to the presence of specific chemicals that bind to

the enzyme. When the binding of the chemical shuts off enzyme activity, the process is called

_________ and the chemical is called an _________ .

(a) activation, activator

(b) inhibition, inhibitor

(c) Inhibition, promoter

(d) activation, inhibitor

Inhibition of succinate dehydrogenase by malonate is an example of

(a) Non-competitive inhibition

(b) Negative feed back

(c) Allosteric inhibition

(d) Competitive inhibition

Which one of the following statement is incorrect?

(a) A competitive inhibitor reacts reversibly with the enzyme to form an enzyme inhibitor complex

(b) In competitive inhibition, the inhibitor molecule is not chemically change by the enzyme

(c) The competitive inhibitor does not affect the rate of breakdown of the enzyme subtract complex

(d) The present of the competitive inhibitor decreases the Km of the enzyme for the substrate.

Suicidal Enzyme is:

a. Lipoxygenase

b. Cyclooxygenase

c. Thromboxane Synthase

d. 5’ Nucleotidase

Noncompetitive enzyme inhibition leads to:

a. Vmax ↑

b. Vmax ↓

c. Vmax unchanged

d. Km ↑

e. Km ↓

True about competitive inhibition of enzyme:

a. ↑ Km

b. ↓ Km

c. ↑ Vmax

d. No change in Km and Vmax

e. Vmax remain same

Noncompetitive reversible inhibitors:

a. Raise Km

b. Lower Km

c. Lower Vmax

d. Raise both Vmax and Km.

e. Do not affect either Vmax or Km

Km changes and Vmax remains the same. What is the type of Enzyme inhibition?

a. Competitive Inhibition

b. Noncompetitive Inhibition

c. Uncompetitive inhibition

d. Suicide Inhibition

Chymotrypsinogen is a:

a. Zymogen

b. Carboxypeptidase

c. Transaminase

d. Exopeptidase

Trypsin is a:

a. Serine protease

b. Lecithinase

c. Phospholipase

d. Elastase