Lec 11: Enzyme Mechanisms Study Notes

Enzyme Mechanisms Learning Goals

  • Understanding how the energy of the transition state is lowered.

  • Knowing the role of acid-base catalysis in enzyme function.

  • Analyzing the shapes and characteristics of enzyme active sites.

  • Exploring covalent catalysis, focusing on how intermediates are stabilized.

  • Investigating the effects of electrostatics in catalytic processes.

  • Learning about enzyme inhibitors, specifically irreversible inhibitors.

  • Required Reading: Chapter 6, section 6.4, up to page 208, and textbook problems 21 and 25.

Enzyme Mechanisms Overview

General Properties of Enzymes

  • Enzymes significantly ↑ the rate of reactions (many fold).

  • They exhibit specificity for their substrates.

  • Function efficiently under physiological conditions.

Active Sites

  • Enzymes have elaborate active sites which bind substrates specifically due to favorable molecular interactions.

  • The enzyme-substrate (ES) complex is stable—substrates are bound such that they are favorably predisposed for product formation.

  • The transition state is stabilized through orientation and proximity of functional groups involved in the reaction.

  • Cofactors may aid in catalysis by stabilizing charge or facilitating interactions.

Types of Enzyme Mechanisms

Mechanisms of Catalysis

  1. Acid-Base Catalysis

    • Protons are transferred, facilitating reactions.

  2. Covalent Catalysis

    • Involves the formation of a transient covalent bond between the enzyme and substrate, resulting in covalently attached intermediates.

  3. Electrostatic Catalysis

    • The stabilization of charged transition states by selectively polarizing and orienting charge through electrostatic interactions.

  4. Metal Ion Catalysis

    • Utilizes metal ions (e.g., Zn²⁺) as electrophiles in reactions.

  5. Proximity and Orientation

    • The substrate is oriented properly and brought into close proximity to the reactive site, facilitating reactions.

  6. Preferential Binding of Transition State

    • The enzyme may preferentially bind the transition state, stabilizing it relative to the reactants.

Amino Acids and Their Role in Catalysis

Functional Groups in Catalysis

  • Catalysis is mediated by functional groups in the 20 amino acids, alongside metals and coenzymes.

  • Key amino acids involved in acid-base catalysis include:

    • Glu (Glutamic acid), Asp (Aspartic acid): Have carboxylic acid groups that can act as proton donors.

    • Lys (Lysine), Arg (Arginine): Positively charged amines that can accept protons.

    • Cys (Cysteine): Contains a thiol group (-SH) that can participate in nucleophilic attacks.

    • His (Histidine): Can function as both a proton donor and acceptor depending on the protonation state.

    • Ser (Serine), Tyr (Tyrosine): Contains polar hydroxyl (-OH) groups.

Nucleophiles and Electrophiles

Nucleophiles

  • Negatively charged species that can donate electrons:

    • Oxygen with negative charge (e.g., unprotonated hydroxyl, ionized carboxylic acid).

    • Negatively charged sulfhydryl groups (R-S⁻).

    • Carbanions (C⁻) and uncharged amines (R-NH₂).

Electrophiles

  • Positively charged or electron-deficient species that can accept electrons:

    • Carbon atoms of carbonyl groups; the more electronegative oxygen pulls electrons away, facilitating reactivity.

    • Protonated imine groups can activate for nucleophilic attack.

    • Phosphorus in phosphate groups can serve as electrophiles.

Proteases: Enzymes that Cleave Peptide Bonds

  • Proteases cleave proteins by hydrolysis—a reaction that is exergonic but kinetically slow due to the resonance stabilization of the peptide bond, which contributes to its resistance to hydrolysis.

Specific Types of Proteases

  • Trypsin: Cleaves on the carboxyl side of Lysine (Lys) and Arginine (Arg).

  • Chymotrypsin: Cleaves on the carboxyl side of aromatic amino acids.

  • Elastase: Cleaves on the carboxyl side of small R groups.

Serine Proteases

General Characteristics

  • Serine acts as a nucleophile at the active site of serine proteases.

  • They utilize a catalytic mechanism that involves:

    • Covalent catalysis.

    • Proximity and orientation effects.

    • General acid-base catalysis.

    • Electrostatic catalysis.

    • Transition state stabilization.

    • Regulation and control of catalysis.

Chymotrypsin Catalytic Site

  • Comprises three residues:

    • His 57 (H),

    • Asp 102 (D),

    • Ser 195 (S).

  • Known as the catalytic triad, which is essential for the mechanism and catalytic activity, stabilized by favorable interactions among these groups.

Mechanism of Chymotrypsin

Overall Mechanism Steps

  1. Substrate Binding:

    • The substrate binds to the active site such that the bond to be cleaved (scissile bond) is in proximity to the catalytic serine (Ser195).

  2. Nucleophilic Attack:

    • The serine's alkoxide ion attacks the carbonyl carbon of the substrate. This is facilitated by His57, which acts as a base by deprotonating Ser195.

  3. Formation of Tetrahedral Intermediate:

    • A short-lived tetrahedral intermediate is formed, stabilized by the oxyanion hole.

  4. Collapse of the Intermediate:

    • The tetrahedral intermediate collapses, leading to the release of the first product and the formation of the acyl-enzyme intermediate, facilitated by protonation of the leaving group by His57.

  5. Water Molecule Attack:

    • A water molecule is deprotonated by His57 to generate hydroxide ion, which attacks the acyl-enzyme complex.

  6. Second Tetrahedral Intermediate:

    • The second tetrahedral intermediate is stabilized by the oxyanion hole.

  7. Final Collapse:

    • The intermediate collapses, leading to the release of the second product and restoration of the free enzyme.

Enzyme Inhibition

Irreversible Inhibition in Serine Proteases

  • All serine proteases possess a “special Ser” residue critical for their nucleophilic activity.

  • The environment surrounding this Ser residue makes it uniquely reactive compared to other serines in the enzyme, which do not undergo similar reactivity.

Examples of Acetylcholinesterase Inhibitors

  1. Acetylcholine: Hydrolysis results in the production of choline.

    • Equation: (CH₃)₃N-CH₂-CH₂-O-C-CH₃ + H₂O → (CH₃)₃N-CH₂-CH₂-OH + Choline.

  2. Parathion and Malathion: Organophosphate compounds that inhibit acetylcholinesterase.

Further Understanding of Enzyme Mechanisms

  • Enzymes not only speed up reactions but also ensure specificity and efficiency under physiological conditions, relying on detailed interactions between substrates and the active site.

  • Understanding transition states and mechanisms deepens the insight into enzyme kinetics and potential applications in drug design and biotechnology.