Mechanisms and Inhibitors

Chapter 9: Mechanisms and Inhibitors

Transition-State Formation

• Enzymes catalyze a vast array of chemical reactions, facilitating the formation of the transition state.

• Transition state formation varies among enzymes, but common strategies include:

  • Covalent catalysis

  • General acid–base catalysis

  • Metal ion catalysis

  • Catalysis by approximation

Covalent Catalysis

• In covalent catalysis, the active site contains a reactive group (usually a powerful nucleophile) that becomes temporarily covalently modified during catalysis.

  • Nucleophile:

  • Definition: A chemical group or atom attracting regions of positive charge in other molecules, donating electrons to electrophiles.

  • Electrophile:

  • Definition: An electron-deficient chemical group that attracts negative charge, accepting electrons from nucleophiles.

General Acid–Base Catalysis

• In this type of catalysis, a molecule other than water acts as a proton donor or acceptor.

  • Example: Chymotrypsin utilizes a histidine residue as a base catalyst to enhance the nucleophilic power of serine.

Metal Ion Catalysis

• Metal ions can act catalytically in numerous ways:

  • As electrophilic catalysts stabilizing a negative charge on reaction intermediates.

  • Generating nucleophiles by increasing the acidity of nearby molecules (e.g., water).

  • Binding to substrates, increasing interactions with the enzyme and thus binding energy.

  • Serving as cofactors for various enzymes.

Catalysis by Approximation

• Many reactions involve two distinct substrates.

  • Reaction rates can be significantly increased by positioning both substrates in proximity and proper orientation on a single binding surface of an enzyme.

Binding Energy

• Binding energy is maximized when the enzyme binds to the transition state, which favors its formation and promotes catalysis.

Temperature Effects on Enzyme Activity

• Generally, as temperature rises, the rate of most reactions increases due to greater molecular motion (Brownian motion).

• Endotherms (e.g., humans) maintain constant body temperatures:

  • Less sensitivity to ambient temperature, but tissue temperature changes can impact enzyme activity (e.g., athlete muscle warming).

• Ectotherms:

  • Organisms that assume ambient environmental temperature.

  • Example: Lizards are more active in warmer temperatures.

  • Thermophilic archaea can thrive at temperatures of 80°C or higher, with proteins adapted to resist thermal denaturation.

Enzyme pH Dependence

• Enzyme activity varies with [H+], typically showing a bell-shaped curve concerning pH.

• Optimal pH varies between enzymes corresponding to their environments:

  • Pepsin: Functions in the stomach at pH 1-2.

  • Chymotrypsin: Optimal near pH 8 in the pancreatic secretion of the upper small intestine.

Ionizable R Groups and pH Dependence

• Activity curves depend on multiple ionizable groups:

  • For example, glutamic acid and lysine residues in an enzyme may vary in activity with changing pH.

  • Changes in pH affect the ionization states of these residues, impacting enzyme activity.

Enzyme Inhibition

• Enzyme inhibition is a major biological control mechanism, often regulating allosteric enzymes.

• Inhibition can be caused by small molecules and ions, with implications for drug design and toxicology.

  • Inhibitors can reveal insights into enzyme mechanisms, often identifying residues crucial for catalysis.

Types of Inhibition

• Irreversible Inhibition:

  • Inhibitors covalently bind to their target enzymes and do not dissociate appreciably.

  • Examples:

  • Penicillin: Covalently modifies transpeptidase, blocking bacterial cell wall synthesis.

  • Aspirin: Covalently modifies cyclooxygenase to reduce pro-inflammatory signals.

• Reversible Inhibition:

  • Characterized by the dissociation of the enzyme–inhibitor complex via noncovalent interactions.

  • Types:

  • Competitive Inhibition:

  • Inhibitor competes with substrate for binding:

    • Example: Sulfanilamide (antibacterial agent).

  • Uncompetitive Inhibition:

  • Inhibitor binds to the enzyme–substrate complex, inhibiting catalysis without preventing substrate binding.

  • Noncompetitive Inhibition:

  • Inhibitor can bind to both free enzyme and enzyme–substrate complex, reducing catalysis without dependence on substrate concentration.

Reversible Inhibition Kinetics

• Competitive Inhibition Kinetics:

  • Inhibitor dissociation constant: $K_i = rac{[E][I]}{[EI]}$.

  • Smaller $K_i$ indicates more potent inhibition.

  • Can be overcome by high substrate concentration, increasing the apparent $K<em>M$ to $K</em>{Mapp}$.

  • The y-intercept (1/Vmax) remains constant, while slope (KM/V_max) increases with inhibition.

• Uncompetitive Inhibition Kinetics:

  • Inhibitor binds only to the enzyme-substrate complex (ES), lowering $V<em>{max}$ to $V</em>{max app}$ and decreasing the apparent $K<em>M$ to $K</em>{Mapp}$.

  • The slope of the line remains consistent; the y-intercept increases.

• Pure Noncompetitive Inhibition Kinetics:

  • $K<em>M$ remains unchanged, while $V</em>{max}$ decreases.

  • The y-intercept and slope values increase by the same factor.

• Mixed Noncompetitive Inhibition Kinetics:

  • Inhibitor preferentially binds to either free enzyme or enzyme–substrate complex, affecting both $K<em>M$ and $V</em>{max}$.

Inhibitor Identification

• Possible pathways:

  • No inhibition?

  • Determine type of inhibition:

  1. Competitive

  2. Uncompetitive

  3. Noncompetitive

Transition-State Analogs

• Proposed by Linus Pauling in 1948 to be effective inhibitors.

• Highly potent competitive inhibitors created by synthesizing compounds that resemble transition states closely.

• Best structural similarity translates to better binding and supportive evidence for transition-state structure hypotheses.

Group-Specific Reagents

• Irreversible inhibitors modifying specific R groups of amino acids:

  • Example:

  • Diisopropylphosphofluoridate (DIPF): Inhibits chymotrypsin by modifying a reactive serine residue.

Affinity Labels (Reactive Substrate Analogs)

• Inhibit enzymes by being similar to substrates, forming covalent bonds with active site residues.

  • Example:

  • Tosyl-L-phenylalanine chloromethyl ketone (TPCK): Irreversibly modifies histidine in chymotrypsin.

Mechanism-Based (Suicide) Inhibitors

• These inhibitors are chemically modified substrates that act as specific means of modifying an enzyme's active site.

  • They bind to the enzyme as a substrate and participate in the catalytic mechanism, generating a reactive intermediate that covalently modifies the enzyme.

• Example: Penicillin is a suicide inhibitor for bacterial cell wall synthesis.

Clinical Insight: Penicillin

• Structure: A thiazolidine ring fused to a β-lactam ring with a variable R group.

• Mechanism: Interferes with bacterial cell wall synthesis by inactivating transpeptidase, which creates cross-links in peptidoglycan.

  • Penicillin mimics D-Ala-D-Ala of the substrate, forming a covalent bond with serine at the active site.

Protein Turnover

• Important biological process for recycling amino acids after protein degradation.

• Proteolytic enzymes (proteases) catalyze protein breakdown via hydrolysis reactions.

Chymotrypsin as a Protein-Degrading Enzyme

• Cleaves peptide bonds to the carboxyl-terminal side of large hydrophobic amino acids (e.g. tryptophan, tyrosine).

  • Uses covalent modification as its catalytic strategy.

Monitoring Chymotrypsin Activity

• Kinetics monitored using chromogenic substrate analogs that form colored products.

  • E.g., N-acetyl-L-phenylalanine p-nitrophenyl ester generates yellow p-nitrophenolate, measurable via absorbance.

Chymotrypsin Kinetics

• Exhibits Michaelis–Menten kinetics under steady-state conditions, producing a burst of product followed by slower formation.

Chymotrypsin Action: Two-Step Mechanism

• Enzyme cleaves substrate in two steps, forming an acyl-enzyme intermediate. - The first step is rapid; the second is slower (resetting the enzyme).

Affinity Labeling and Histidine 57 Role

• Histidine 57's role was demonstrated via affinity labeling using TPCK, which binds and modifies the histidine, rendering chymotrypsin inactive.

Catalytic Triad Components

• The catalytic triad comprises:

  • Serine 195

  • Histidine 57

  • Aspartate 102

• The triad facilitates the deprotonation of serine by histidine, providing a powerful nucleophile.

Mechanism for Peptide Hydrolysis

• Steps include:

1. Substrate binding

2. Nucleophilic attack by serine on carbonyl carbon (tetrahedral intermediate formation)

3. Stabilization of the tetrahedral intermediate via the oxyanion hole

4. Collapse to generate the acyl-enzyme intermediate

5. Deacylation, where water replaces the amine group

6. Hydrolysis of the ester in a repeated process

S1 Pocket Function

• The S1 pocket accommodates large, uncharged side chains (e.g., phenylalanine, tryptophan), positioning peptide bonds for cleavage.

Catalytic Triad Amino Acids

• Aspartate 102

• Histidine 57

• Serine 195

Additional Questions:

1. Which amino acids are in the catalytic triad?

   • Answer: Aspartate 102, Histidine 57, Serine 195

2. Which amino acids are in the catalytic triad?

   • Answer: Aspartate 102, Histidine 57, Serine 195