Enzyme Inhibition Notes

Enzyme Inhibition

Learning Goals

• 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 Alteration

• 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).

Allosteric Regulation of Enzymes

• 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.

Types of Enzyme Inhibition

• 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.

Competitive Inhibition

• 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.

Effect on Kinetics

• 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).

Non-Competitive Inhibition

• 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.

Effect on Kinetics

• 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.

Uncompetitive Inhibition

• 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.

Effect on Kinetics

• 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.

Mixed Inhibition

• 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.

Irreversible Inhibition

• 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).

Allosteric Activators

• 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.

Summary

• 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).