Sequence in which enzyme-bound reaction intermediates form
Structures of each intermediate and transition state
Rate of interconversion to each intermediate
Structural relationship between the enzyme and each intermediate
Energy contributions of all reacting and interacting transition states and complexes
Note: Very few enzymes are well-characterized to this level of detail.
Model Enzyme Reaction Mechanisms
Well-Studied Enzymes
Chymotrypsin
Hexokinase
Enolase
Human Chymotrypsin
Structure referenced (PDB coordinates: 4H4F).
Bovine Pancreatic Chymotrypsin
Classification: Protease (enzymes that hydrolyze the peptide bond)
Molecular Weight (MW): 25,191 Da
Specificity:
Preferred cleavage adjacent to W (tryptophan), F (phenylalanine), Y (tyrosine) amino acids
Key features:
Illustrates transition-state stabilization
Involves general acid-base chemistry
Employs covalent catalysis
Chymotrypsin Structure
Primary Structure:
2-chain protease with inter- and intra-disulfide linkages (between Cys amino acids)
Active site amino acids highlighted (red) perform catalysis
Surface Representation:
Red patches indicate active site residues responsible for catalysis
Yellow hydrophobic pocket shows preference for aromatic containing peptide cleavage
Ribbon Representation:
Displays disulfide linkages (yellow) and R-groups of catalytic amino acids
Mechanism of Chymotrypsin Catalysis
Enhanced the rate of peptide bond hydrolysis by a factor of at least 10^9.
Distinct Mechanism Phases:
Acylation Phase
Deacylation Phase
Kinetics and Catalysis
Chymotrypsin can hydrolyze small esters and amides slower than polypeptides due to less binding energy available.
Hartley and Kilby study used p-nitrophenylacetate substrate:
Hydrolysis produces yellow color (p-nitrophenol) that can be monitored at 405 nm.
Kinetic analysis showed a burst of product followed by a slower phase, indicating that the rate-limiting step is the regeneration of the enzyme.
pH Influence on Enzyme Activity
Enzyme-catalyzed reactions are pH-sensitive, each enzyme having an optimal pH for catalysis: Chymotrypsin optimum pH = 8.
Importance of pH Effects:
What happens to the R-group of histidine at low pH?
High pH (pH > 10) leads to the stabilization of the enzyme’s active conformation via salt bridges involving Asp194 and Ile16.
Loss of salt-bridge interaction (e.g., at pH 12) disrupts enzyme activity.
Review of Histidine pKa
Catalytic triad components:
Asp102 (D102), His57 (H57), Ser195 (S195)
Shift of His57 pKa:
From a typical value (6) to higher (12) in chymotrypsin, allowing it to act as a general base to remove a proton from Ser195, enhancing nucleophilic power of Ser195.
Chymotrypsin Reaction Mechanism (Detailed Steps)
Formation of Tetrahedral Intermediate:
Oxygen from Ser195 attacks the carbonyl of the substrate, resulting in a temporary charge development stabilized by the oxyanion hole.
Hydrogen bonds from enzyme backbone reduce energy for transition state formation.
Release of Product 1:
Collapse of intermediate regenerates double bond and protonation of the amino group by His57, releasing the first peptide fragment.
Role of Water in Deacylation:
Water acts as the second substrate.
His57 deprotonates water, creating a second tetrahedral intermediate.
Final Tetrahedral Intermediate Collapse:
Formation of the second product (carboxylate anion) displaces Ser195, regenerating the active enzyme state for re-catalysis.
Summary of Reaction Steps
Steps 1-4 involve the acylation of Ser195 with the release of product 1 (covalent catalysis) and transition-state stabilization through the oxyanion hole.
Deacylation steps release product 2 and regenerate the enzyme.
His57’s role as a general base is crucial to facilitate these reactions.
Alanine Scanning Mutagenesis
A technique to identify critical amino acids in enzymes:
Mutate amino acids to alanine, assessing resulting activity.
Focus typically on specific regions of the enzyme.
Why Alanine?: Alanine’s simple side chain is less likely to perturb protein structure compared to other mutations.
Protease Classes
Four distinct classes based on catalytic mechanism:
Serine Proteases (e.g., Chymotrypsin)
Cysteine Proteases
Aspartate Proteases
Metalloproteases
Conformational Change in Enzyme Catalysis
Importance of conformational changes in facilitating catalysis.
Hexokinase
Catalyzes reversible phosphorylation of glucose:
Adds phosphate to the hydroxyl group at C6 of glucose.
Induced Fit Model:
Hydroxyl group of glucose has reactivity similar to water.
Water enters the active site without being phosphorylated.
Hexokinase Mechanism
2-Substrate Kinase (bisubstrate):
Inactive without substrate; active when substrates bind, prompting conformational shifts positioning crucial amino acids for catalysis.
Xylose and Activity:
Similar to glucose but shorter; can bind but doesn’t position hydroxyl for phosphorylation, leading to ATP hydrolysis without productive reaction.
Metal-Ion Chemistry in Enzyme Catalysis
Analysis of the role of metal ions in catalytic processes.
Enolase
Catalyzes dehydration of 2-Phosphoglycerate to phosphoenolpyruvate and vice versa.
Emphasizes the use of enzymatic cofactors in catalysis:
Involves metal-ion catalysis, general acid-base chemistry, and transition-state stabilization.
Enolase Active Site
Key residues in the active site:
Lys345 as general base (removing H+ from substrate)
Glu211 donates a proton to leaving -OH group.
Mg2+ enhances ionic interactions and lowers pKa for carboxyl group, facilitating proton abstraction.
Key Points to Understand
Mechanisms of chymotrypsin catalysis, acid-base chemistry, and the significance of the oxyanion hole.
Pre-steady state kinetics indicating deacylation as a rate-limiting step.
Effects of pH on enzyme reactions, particularly regarding amino acid pKa.
Utilization of alanine in mutagenesis versus glycine.
Overview of protease classes and importance of Ixhibiters concerning HIV protease.
Significance of Mg2+ ions in the function of enolase.