BIOCHEM2027 Specialised Subject · 4.4
2027 · High School Specialised Subject · Biochemistry

6.4 Examples of Enzyme Reactions

Enzyme mechanisms: six case studies

Acylation and deacylation in chymotrypsin, HIV protease and mechanism-based drugs, induced fit in hexokinase, metal catalysis in enolase, lysozyme, and PBPs with beta-lactams.

7 SECTIONS20 FIGURESEnglish edition
01

Acylation and deacylation in chymotrypsin

  • A serine protease preferring peptide bonds on the carboxyl side of Phe, Tyr and Trp, with a hydrophobic S1 pocket for selectivity
  • Catalytic triad Ser195-His57-Asp102: His57 acts as general acid and base, Asp102 sets charge and orientation, Ser195 attacks nucleophilically
  • The oxyanion hole formed by backbone NH groups stabilises the negative charge of the tetrahedral intermediate
  • Acylation: first tetrahedral intermediate, peptide cleavage and amine departure, then the acyl-enzyme
  • Deacylation: water attacks the acyl-enzyme, a second tetrahedral intermediate collapses, acid product leaves and free enzyme returns
  • A burst followed by a slow steady state with ester substrates suggests but does not prove rate-limiting deacylation
Figure 6.4-63Catalytic reactions of six different enzymes and the principles they share
Figure 6.4-64Four core questions used to compare enzyme mechanisms
Figure 6.4-65S1 binding pocket of chymotrypsin and substrate selectivity
Figure 6.4-66The Asp102-His57-Ser195 triad and the role of the oxyanion hole
Figure 6.4-67Catalytic cycle from substrate binding to release of both products
Figure 6.4-68Model energy curve separating the transition state from the acyl-enzyme intermediate
Figure 6.4-69Simulated progress curve showing the burst phase
02

HIV protease and mechanism-based drugs

  • An aspartic protease that cleaves Gag and Gag-Pol polyproteins to allow viral maturation
  • A symmetric dimer whose Asp25 and Asp25-prime form the catalytic apparatus, using water as nucleophile rather than a serine acyl-enzyme
  • Inhibitors mimic the high-energy state of cleavage with a non-cleavable structure that binds the active site tightly
  • Blocked polyprotein processing lowers the infectivity of new particles, combination therapy matters and the drugs do not eliminate HIV
  • IC50, the concentration giving 50 percent inhibition under defined conditions, is not automatically equal to Ki
  • Example: IC50 2.0 micromolar with inhibitor at 6.0 micromolar gives v/v0 = 1/(1+3) = 0.25, so 25 percent activity remains
Figure 6.4-70Active site formed jointly by Asp25 and Asp25-prime of the dimer
Figure 6.4-71How substrate cleavage and inhibitor occupancy differ in their effect on viral maturation
Figure 6.4-72Residual activity curves for two hypothetical inhibitors with different IC50 values
03

Substrate binding and induced fit in hexokinase

  • Phosphorylation at carbon 6 of glucose, with MgATP as the usual phosphate donor
  • Glucose binding narrows the cleft between two domains, aligns the substrate and limits water access, suppressing wasteful ATP hydrolysis
  • About 12 degrees of domain rotation on glucose binding was reported for yeast hexokinase
  • Induced fit emphasises flexible structural change, but selectivity also depends on affinity, active-site chemistry and the energy landscape
  • Observing a structural change does not by itself quantify the catalytic effect of a step
Figure 6.4-73Relative domain motion before and after glucose binding
Figure 6.4-74Phosphate transfer to the C6 hydroxyl of glucose and the role of MgATP
04

Metal ion catalysis in enolase

  • A reversible dehydratase (EC 4.2.1.11) that removes water from 2-phosphoglycerate to give phosphoenolpyruvate
  • Pyruvate kinase uses the PEP phosphate to make ATP in the next step, but the essence here is dehydration rather than ATP synthesis
  • Mg2+ coordinates substrate oxygens and stabilises the anionic intermediate while active-site residues assist proton transfer
  • Too little Mg2+ limits activity, and very high levels can lower it through extra binding or hindered product release
  • Records must state the metal species, free Mg2+ concentration, pH and substrate concentration
Figure 6.4-75Reversible conversion of 2-PG to PEP and water
Figure 6.4-76Model of Mg2+ coordinating the substrate and stabilising charge
Figure 6.4-77Simulated curve showing activation at low ion concentration and inhibition at high concentration
05

Glycosidic cleavage by lysozyme

  • Found in tears, saliva and egg white, hydrolysing the beta(1-4) glycosidic bond between NAM and NAG in peptidoglycan
  • In hen egg-white lysozyme Glu35 acts as acid and base while Asp52 stabilises charge or forms a covalent intermediate
  • The standard model is two successive nucleophilic substitutions, with a sugar-enzyme intermediate formed first and then broken by water
  • That route explains retention of configuration at the anomeric carbon, though an ideal SN2 transition state is not established for every step
  • Reports of a covalent intermediate coexist with objections from hydrogen-bond networks, so textbook arrows must be separated from evidence
Figure 6.4-78Repeating NAG and NAM structure and the bond cleaved by lysozyme
Figure 6.4-79Teaching two-step model showing the roles of Glu35 and Asp52
Figure 6.4-80Covalent-intermediate evidence compared with an alternative structural interpretation
06

Mechanistic insight and antibiotic development

  • Penicillin-binding proteins are the peptidoglycan cross-linking enzymes, with a serine transpeptidase acting on the D-Ala-D-Ala end
  • Beta-lactams react with the active-site Ser of PBPs to form a long-lived acyl-enzyme that blocks the normal cycle
  • Resistance can arise from beta-lactamase hydrolysis, altered target PBPs, changed membrane permeability or drug efflux
  • Tight binding alone does not make a good drug, since access, selectivity, toxicity, metabolic stability and resistance all matter
표 6.4-2Nucleophile and acyl-enzyme step in three enzymes
Figure 6.4-81Formation of peptidoglycan cross-links by a penicillin-binding protein
Figure 6.4-82Acylation and inactivation of the catalytic serine of a PBP by a beta-lactam
07

Synthesis across the six mechanisms

  • Shared features: the active site aligns substrate, stabilises the transition state selectively and regenerates after each cycle
  • The nucleophile differs: serine hydroxyl in chymotrypsin and PBPs, catalytic water in HIV protease, metal-assisted water in enolase
  • Presence or absence of an acyl-enzyme step is a key discriminator, though sharing it does not make mechanisms identical
  • Covalent intermediates and transition states are different, and independent evidence such as bursts, structures and inhibitor complexes must agree
표 6.4-3Key evidence and common confusions for the six mechanisms
2027 High School Specialised Subject · Biochemistry · Sangil Park (Asan High School) · Reference material is printed as an appendix.
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