3 ezyme inhibition and catalysis

What is enzyme inhibition?

A reduction in enzyme activity caused by a molecule binding to the enzyme.

Why is enzyme inhibition important?

It regulates metabolic pathways, explains drug action, and helps study enzyme mechanisms.

What are the two main classes of enzyme inhibition?

Irreversible inhibition and reversible inhibition.

What is irreversible inhibition?

Inhibitor forms a permanent covalent bond with the enzyme, permanently inactivating it.

How is irreversible inhibition reversed?

Only by synthesising new enzyme protein.

Why are irreversible inhibitors often used as drugs?

Because they produce prolonged enzyme inhibition.

Give examples of irreversible enzyme inhibitor drugs.

Aspirin, penicillin, ibrutinib, acalabrutinib.

How does penicillin inhibit enzymes?

It covalently inhibits bacterial transpeptidase, preventing cell wall synthesis.

How does aspirin inhibit cyclooxygenase?

It covalently binds a reactive serine residue in COX, irreversibly inhibiting prostaglandin synthesis.

Why does aspirin have antiplatelet effects?

It blocks thromboxane A₂ production in platelets, reducing clot formation.

What is Bruton’s tyrosine kinase (BTK)?

An enzyme involved in B-cell activation.

How does ibrutinib work?

It irreversibly inhibits BTK by covalent binding to a cysteine residue.

What diseases is ibrutinib used for?

B-cell cancers such as CLL, MCL, and DLBCL.

What is reversible inhibition?

Inhibitor binds non-covalently and inhibition can be reversed.

What are the major reversible inhibition types?

Competitive, non-competitive, uncompetitive, mixed.

What is competitive inhibition?

Inhibitor competes with substrate for the active site.

Where does a competitive inhibitor bind?

The active site.

Can substrate and competitive inhibitor bind simultaneously?

No.

How can competitive inhibition be overcome?

By increasing substrate concentration.

Effect of competitive inhibition on Km?

Km increases.

Effect of competitive inhibition on Vmax?

Vmax unchanged.

Why does Km increase in competitive inhibition?

More substrate is needed to reach half maximal velocity.

Why does Vmax stay unchanged in competitive inhibition?

Because enough substrate can outcompete the inhibitor.

Lineweaver-Burk pattern for competitive inhibition?

Lines intersect at the y-axis.

Example of competitive inhibition drug?

Methotrexate.

How does methotrexate work?

It competes with dihydrofolate for dihydrofolate reductase binding.

Why is methotrexate effective in cancer?

It inhibits DNA synthesis in rapidly dividing cells.

Example of physiological competitive inhibition?

Succinate inhibiting fumarase or malonate inhibiting succinate dehydrogenase.

What is non-competitive inhibition?

Inhibitor binds a separate site and reduces enzyme activity.

Where does a non-competitive inhibitor bind?

An allosteric site, not the active site.

Can substrate and non-competitive inhibitor bind simultaneously?

Yes.

Can increasing substrate overcome non-competitive inhibition?

No.

Effect of non-competitive inhibition on Km?

Km unchanged.

Effect of non-competitive inhibition on Vmax?

Vmax decreases.

Why does Vmax decrease in non-competitive inhibition?

Some enzyme becomes inactive, reducing total catalytic capacity.

Why is Km unchanged in non-competitive inhibition?

Substrate binding affinity remains unchanged.

Lineweaver-Burk pattern for non-competitive inhibition?

Lines intersect at the x-axis.

Example of non-competitive inhibition?

AMP inhibition of fructose 1,6-bisphosphatase.

What is uncompetitive inhibition?

Inhibitor binds only the ES complex.

Can uncompetitive inhibitors bind free enzyme?

No.

Effect of uncompetitive inhibition on Km?

Km decreases.

Effect of uncompetitive inhibition on Vmax?

Vmax decreases.

Why can substrate not overcome uncompetitive inhibition?

Because inhibitor only binds after substrate is bound.

What is mixed inhibition?

Inhibitor binds enzyme and ES complex with different affinities.

Effect of mixed inhibition?

Both Km and Vmax change.

What is Ki?

The inhibitor dissociation/inhibition constant.

What does a low Ki mean?

High inhibitor affinity and high potency.

What does Ki conceptually resemble?

Km for inhibitors.

What does Lineweaver-Burk plotting help identify?

Type of inhibition and kinetic constants.

What is the Michaelis-Menten single substrate model limitation?

Many enzymes use multiple substrates.

What are the two main multi-substrate enzyme mechanisms?

Sequential and double displacement.

What is a sequential reaction mechanism?

Both substrates bind before any product is released.

What is a ternary complex?

Enzyme bound to two substrates simultaneously.

Example of sequential enzyme mechanism?

Lactate dehydrogenase.

Reaction catalysed by lactate dehydrogenase?

Pyruvate + NADH → Lactate + NAD⁺

What is ordered sequential binding?

Substrates bind in a specific order.

Example of ordered sequential enzyme?

Lactate dehydrogenase.

What is random sequential binding?

Substrates bind in any order.

Example of random sequential enzyme?

Creatine kinase.

What is a double displacement reaction?

One substrate binds and one product leaves before second substrate binds.

Alternative name for double displacement?

Ping-pong mechanism.

Is a ternary complex formed in double displacement?

No.

What happens to the enzyme in double displacement?

A temporary modified enzyme intermediate forms.

Example of double displacement enzyme?

Aspartate aminotransferase.

What does aspartate aminotransferase transfer?

An amino group.

How much can enzymes enhance reaction rates?

Up to 10¹⁷-fold, typically around 10¹⁰-fold.

Do enzymes change reaction equilibrium?

No.

What do enzymes change?

The reaction rate.

How do enzymes accelerate reactions?

By lowering activation energy (ΔG‡).

How else do enzymes speed reactions?

By stabilising the transition state.

Why are uncatalysed reactions slow?

High activation energy, unstable charges, poor molecular orientation.

How does the active site help catalysis?

Binds substrate, positions molecules, stabilises transition state, reduces entropy cost.

What interactions bind substrate in the active site?

Hydrogen bonds, hydrophobic interactions, electrostatic interactions, van der Waals forces.

Why does induced fit improve catalysis?

It optimises substrate positioning and transition state stabilisation.

Why are enzymes highly specific?

Because active site shape and charge complement specific substrates.

What is chymotrypsin?

A digestive protease in the intestine.

What reaction does chymotrypsin catalyse?

Hydrolysis of peptide bonds.

What amino acids does chymotrypsin preferentially cleave after?

Aromatic amino acids: phenylalanine, tyrosine, tryptophan.

Does chymotrypsin cleave all peptide bonds equally?

No, specificity depends on side chain compatibility.

Why is chymotrypsin highly specific?

Its active site contains a hydrophobic pocket complementary to aromatic residues.

What is transition state stabilisation?

Enzyme binding lowers energy of the unstable intermediate state.

Incorrect statement: enzymes increase activation energy?

False—enzymes decrease activation energy.

Do enzymes bind substrate covalently in general catalysis?

Usually no, substrate binding is mainly non-covalent.

What is the biological importance of enzyme inhibition?

Pathway regulation, pharmacology, signalling control, experimental investigation.