Wk 9.3 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 work and describe in detail how enzyme kinetics can be changed by these inhibitors (i.e. effects on Vmax $and$ 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.
Two types:
- Activators: increase activity (e.g., cofactors, fructose 2,6-bisphosphate activates phosphofructokinase 1 which increases glycolysis in response to insulin).
- Inhibitors: decrease activity (e.g., feedback inhibition, herbicides, drugs).
Inhibitors can either 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.
- 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 inhibitor types is based on enzyme kinetics.

Inhibitor competes with the substrate for the binding site.
- Cannot bind at the same time.
- Binds to the free enzyme, not the enzyme-substrate complex.
The inhibitor can bind to the enzyme’s active site.
The design resembles a real substrate or co-factor.

Less substrate can bind.
- The reaction rate decreases at lower substrate concentrations.
- More inhibitor = lower reaction rate.
Increasing substrate concentration decreases the effectiveness of the inhibitor.
$V_{max}$ = unchanged (can still reach the same maximum rate).
$K_M$ = 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 the enzyme-substrate complex.
Renders the enzyme catalytically inactive.
- Prevents product formation (i.e., does not prevent binding » prevents catalysis).

Reduces the effectiveness of both the free enzyme and the enzyme-substrate complex (i.e., the enzyme can’t function as well).
$V_{max}$ = decreases.
- The enzyme is not working as efficiently.
- A subset will always be bound to the inhibitor, so $V_{max}$ decreases.
- The inhibitor cannot be removed by increased substrate concentration.
$K_M$ = unchanged.
- Binds to both the free enzyme and the enzyme-substrate complex.
- Does not change the apparent binding of the enzyme for the substrate.
- Lowers the concentration of usable enzymes.
E.g., Nifedipine (anti-anginal/anti-hypertensive) affects CYP2C9 (cytochrome P450) enzyme.

The 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.
NB: Does not bind to the active site; will only bind once the substrate has bound.

Reduces the effective concentration of the enzyme-substrate complex.
$V_{max}$ = decreases.
- The enzyme-substrate complex does not dissociate.
- The product is not formed, so the reaction rate is decreased.
$K_M$ = decreases.
- Binding efficiency increases.
- The enzyme-substrate complex does not dissociate.
Works best at high substrate concentrations.
E.g., Lithium (antidepressants/bi-polar) affects Inositol monophosphatase.

Resembles non-competitive inhibition (binds at the allosteric site) - binds both before and after substrate binding.
Unlike non-competitive inhibition (equal affinity for free enzyme or enzyme-substrate complex), mixed inhibition 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).
E.g., xanthine oxidase ( $Pd^{2+}$ ) » gout.

Permanently inactivate the enzyme (decreases 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 (a.k.a. affinity label):
- 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 the normal enzymatic reaction, a covalent bond is formed, which results in permanent inactivation (e.g., Penicillin and Aspirin).

Rare but can happen.
Bind to the enzyme and change its shape to increase affinity for the substrate.
E.g., 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 affinity for glucose.
Reference: Zorn & Wells. 2010. Nat Chem Biol.

Q: Which of the following statements about non-competitive inhibitors is CORRECT?
- A. $K_m$ is unchanged
- B. $V_{max}$ is unchanged
- C. Binds to the enzyme-substrate complex only
- D. Binds irreversibly

Q: In enzyme kinetics, the $K_M$ is…
- A. Half $V_{max}$
- B. Unchanged by a competitive inhibitor
- C. Increased by an uncompetitive inhibitor
- D. The negative reciprocal of the x-intercept on a Lineweaver-Burk plot.

Type of Inhibition	Binds to Free Enzyme?	Binds to Enzyme-Substrate Complex?	Effect on $V_{max}$	Effect on $K_M$
Competitive inhibitor	Yes	No	Unchanged	Increase
Non-competitive inhibitor	Yes	Yes	Decrease	Unchanged
Uncompetitive inhibitor	No	Yes	Decrease	Decrease
Mixed inhibitor	Yes	Yes	Decrease	Increase or Decrease

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).