Enzyme Inhibition Notes

Enzyme Inhibition

Understand allosteric regulation, allosteric sites, and allosteric regulators (activators/inhibitors).
Understand which domains in the enzyme can be the target of inhibitors, and the different modes of interaction of inhibitors with enzymes (reversible and irreversible inhibition).
Understand how competitive, uncompetitive, non-competitive, and mixed inhibition works and describe in detail how enzyme kinetics can be changed by these inhibitors (i.e. effects on V{max} and KM).
Understand how irreversible inhibitors, including group-specific inhibitors, reactive substrate analogues, and mechanism-based inhibitors work.

Enzyme activity can be altered by other molecules.
- Activators: Increase enzyme activity (e.g., cofactors, fructose 2,6-bisphosphate activates phosphofructokinase 1, increasing glycolysis in response to insulin).
- Inhibitors: Decrease enzyme activity (e.g., feedback inhibition, herbicides, drugs).
Inhibitors can bind directly to the active site or cause a conformational change in the enzyme (allosteric regulator).

Typically involves a multimer (but not always).
Each subunit has a binding site for the substrate and a separate binding site for the allosteric regulator (allosteric site).
Allosteric Inhibitor: Blocks substrate binding.
Allosteric Activator: Permits substrate binding.
Inhibitors are often used as drugs.

Reversible Inhibitors: Inhibitors bind to enzymes non-covalently.
- Competitive inhibitors
- Non-competitive inhibitors
- Uncompetitive inhibitors
- Mixed inhibitors
Irreversible Inhibitors: Inhibitors bind to enzymes covalently.
- Group-specific inhibitors
- Reactive substrate analogues
- Mechanism-based inhibitors
Differentiation of types of inhibitors is based on enzyme kinetics.

Inhibitor competes with substrate for the binding site.
- Cannot bind at the same time.
- Binds to the free enzyme, not the enzyme-substrate complex.
Inhibitor can bind to the enzyme’s active site.
Design resembles the real substrate or cofactor.

Less substrate can bind, decreasing the reaction rate at lower substrate concentrations.
More inhibitor leads to a lower reaction rate.
Increasing substrate concentration decreases the effectiveness of the inhibitor.
V_{max} is unchanged because the same maximum can still be reached.
K_M increases because the inhibitor interferes with binding.
More substrate is needed to reach V_{max}.
Examples: Methotrexate (cancer), Relenza (influenza).

Binds whether the substrate is bound or not (allosteric site).
- Equal affinity for the free enzyme or enzyme-substrate complex.
Renders the enzyme catalytically inactive.
- Prevents product formation but does not prevent binding.

Reduces the effectiveness of both the free enzyme and the enzyme-substrate complex.
V_{max} decreases because the enzyme is not working as efficiently.
- A subset of enzymes will always be bound to the inhibitor, decreasing V_{max}.
- The inhibitor cannot be removed by increased substrate concentration.
K_M is unchanged.
- Binds to both the free enzyme and the enzyme-substrate complex.
- Does not change the apparent binding of the enzyme for the substrate, as the substrate can still bind after the inhibitor is bound, lowering the concentration of usable enzymes.
Example: Nifedipine (anti-anginal/anti-hypertensive) affects the CYP2C9 (cytochrome P450) enzyme.

Inhibitor binds to the enzyme-substrate complex but not the free enzyme.
The inhibitor-enzyme-substrate complex is catalytically inactive.
- Distorts the active site and prevents product formation.
Does not bind to the active site; will only bind once the substrate has bound.

Reduces the concentration of the effective enzyme-substrate complex.
V_{max} decreases because the enzyme-substrate complex does not dissociate, product is not formed, so the reaction rate is decreased.
K_M decreases because binding efficiency increases and the enzyme-substrate complex does not dissociate.
Works best at high substrate concentrations.
Example: Lithium (antidepressant/bipolar) affects Inositol monophosphatase.

Resembles non-competitive inhibition (binds at the allosteric site) - binds both before and after substrate binding.
Unlike non-competitive inhibition, it does not have equal affinity for the free enzyme or the enzyme-substrate complex, has a greater affinity for one or the other.
V_{max} decreases.
K_M can increase (if it favors binding to the free enzyme) or decrease (if it favors binding to the enzyme-substrate complex).
Example: xanthine oxidase (Pd^{2+}) affects gout.

Permanently inactivates the enzyme, decreasing enzyme concentration.
Group-specific Inhibitors: React with a specific amino acid side chain (e.g., iodoacetamide modifies cysteine residues and inhibits cysteine peptidases).
Reactive Substrate Analogues (Affinity Labels): Structurally similar to the substrate and react with the substrate (e.g., TPCK inhibits chymotrypsin).
Mechanism-based Inhibitors: The inhibitor binds to the active site of the enzyme, so during normal enzymatic reaction, a covalent bond is formed, resulting in permanent inactivation (e.g., Penicillin and Aspirin).

Rare but can happen.
Bind to the enzyme and change its shape to increase its affinity for the substrate.
Example: MK-0941 (Glucokinase activator).
Glucokinase is involved in glycolysis and is inactivated in maturity-onset diabetes of the young (MODY).
MK-0941 allosterically alters the shape of glucokinase and increases its affinity for glucose.

Enzymes can be allosterically regulated - activated or inhibited.
Two types of inhibitors: reversible (competitive, non-competitive, uncompetitive, mixed) and irreversible (reactive substrate analogues, group-specific inhibitors, mechanism-based inhibitors).