Biological context
Secreted by the pancreas as the zymogen chymotrypsinogen; activated by trypsin. Central to protein digestion. Prototype of the S1 peptidase clan — shared architecture with trypsin, elastase, thrombin.
back to catalog
Hydrolase / serine protease · Peptidase S1 (chymotrypsin family)
α-Chymotrypsin
EC EC 3.4.21.1PDB 4CHABos taurus
The paradigm of the catalytic triad (Ser-His-Asp). Two-step mechanism: acylation forms a covalent acyl-enzyme intermediate; deacylation by water releases the C-terminal product.
Mechanistic insight
The oxyanion hole (backbone amides of Gly193 and Ser195) stabilises the tetrahedral transition state by 5–8 kcal/mol — a textbook example of transition-state stabilisation.
serine proteasecatalytic triadcovalentacyl-enzymeoxyanion hole
Catalytic mechanism
Type: Two-step covalent catalysis — acylation / deacylation via catalytic triad
Rate-limiting: Deacylation (for amide substrates) or acylation (for poor leaving groups)
Overall reaction
Peptide-(Phe/Tyr/Trp)-X + H2O → Peptide-(Phe/Tyr/Trp)-OH + H-X-peptide
01
Substrate binding in S1 pocket
An aromatic residue (Phe/Tyr/Trp) of the substrate inserts into the deep hydrophobic S1 pocket (lined by Ser189, Gly216, Gly226). The scissile carbonyl is positioned above Ser195 Oγ, and its oxygen points into the 'oxyanion hole' — the backbone NH groups of Gly193 and Ser195.
A:195A:57A:102A:189A:193
Step 1 / 5
References (3)
- Blow DM, Birktoft JJ, Hartley BS. Role of a buried acid group in the mechanism of action of chymotrypsin. Nature 221:337–340 (1969).
- Polgár L. The catalytic triad of serine peptidases. Cell Mol Life Sci 62:2161–2172 (2005).
- Hedstrom L. Serine protease mechanism and specificity. Chem Rev 102:4501–4524 (2002).
Kinetic parameters
Steady-state kcat, KM and specificity constants from literature.
| Substrate | kcat (s⁻¹) | KM (mM) | kcat/KM (M⁻¹s⁻¹) | pH | T (°C) | Source |
|---|---|---|---|---|---|---|
| Suc-Ala-Ala-Pro-Phe-p-nitroanilide | 63.0 | 0.060 | 1.05 × 10⁶ | 7.9 | 25 | Bauer, Biochemistry 1978 |
| N-Ac-L-Phe-ethyl ester | 28.0 | 0.18 | 1.56 × 10⁵ | 7.9 | 25 | Fersht, Enzyme Structure and Mechanism (reference table) |
| N-Ac-Trp-p-nitrophenyl ester | 18.0 | 0.020 | 9.00 × 10⁵ | 7 | 25 | BRENDA (aggregate) |
Chymotrypsin specificity is ~10⁴-fold higher for aromatic vs aliphatic side chains. For amide substrates deacylation is rate-limiting; for activated esters (p-nitrophenyl) acylation becomes rate-limiting and 'burst kinetics' is observed.
Michaelis–Menten · steady-state simulator
Run Chymotrypsin in silico.
Integrate the reaction v = Vmax·[S] / (Km,app + [S]) in closed form with RK4, across any [S], [E], and an optional reversible inhibitor. Defaults are drawn from the curated kinetic record for this enzyme where available.
Progress curve · [S](t) and [P](t)30 s window
substrate [S]product [P]
Saturation · v vs. [S]Km,app = 0.0600 mM · Vmax,app = 0.0630 mM/s
Inhibitor
Curated kinetic record
Defaults seeded from Suc-Ala-Ala-Pro-Phe-p-nitroanilide at pH 7.9 / 25 °C — source: Bauer, Biochemistry 1978.
Why this enzyme is in the catalog
Gold standard for teaching covalent catalysis, charge-relay, oxyanion hole, serine protease family evolution.