Enzyme catalysis — catalytic triad
Serine protease: catalytic triad mechanism
How the Serine protease works, in 3 steps. Each step shows the structures and the curved arrows that move the electrons.
In short. A 3-step mechanism. Typical reagents: Peptide substrate, chymotrypsin/trypsin-like protease (Ser195, His57, Asp102), H₂O. Conditions: Aqueous, pH ≈ 7–8, 37 °C. Bonds formed: Ser195 acyl-enzyme ester; free carboxylic acid; free amine; bonds broken: Peptide C–N; Ser195 O–H. Stereochemistry: Oxyanion hole (Gly193, Ser195 N–H) stabilises tetrahedral O⁻. Rate and selectivity: Acylation and deacylation both contribute; ~10¹⁰-fold rate enhancement.
At a glance
Step 1: Triad relay activates Ser195
A histidine, positioned by a hydrogen-bonded aspartate, deprotonates the active-site serine's hydroxyl, dramatically boosting its nucleophilicity — the catalytic triad working as a relay to generate an alkoxide-like nucleophile from a normally unreactive alcohol.
Step 2: Attack: tetrahedral intermediate
That activated serine oxygen attacks the substrate's peptide carbonyl carbon — ordinary nucleophilic acyl substitution — forming a tetrahedral intermediate stabilized by backbone N–H hydrogen bonds in the 'oxyanion hole.'
Step 3: Acyl-enzyme, then hydrolysis
Collapse of the intermediate releases the first half of the cleaved peptide and forms a covalent acyl-enzyme intermediate; water (activated by the same histidine) then hydrolyzes this intermediate to release the second half and regenerate the free enzyme.
Abbreviations and terms
- R
- R group. Any carbon substituent (alkyl, aryl…) whose identity does not matter for the mechanism.
- H2
- Hydrogen gas. Reductant for catalytic hydrogenation.
How it compares with related reactions
| Serine protease | Aldolase | DNA polymerase | |
|---|---|---|---|
| Reagents | Peptide substrate, chymotrypsin/trypsin-like protease (Ser195, His57, Asp102), H₂O | Dihydroxyacetone phosphate (DHAP), glyceraldehyde 3-phosphate (G3P), Class I aldolase (Lys229) | Primer–3′-OH, dNTP, 2 Mg²⁺, DNA polymerase (Asp carboxylates) |
| Conditions | Aqueous, pH ≈ 7–8, 37 °C | Aqueous, pH ≈ 7.5 | Aqueous, pH ≈ 7.5, 37 °C |
| Stereochemistry | Oxyanion hole (Gly193, Ser195 N–H) stabilises tetrahedral O⁻ | Enzyme controls the face: gives the D-threo (3S,4R) product | In-line attack, inversion at Pα |
| Rate / selectivity | Acylation and deacylation both contribute; ~10¹⁰-fold rate enhancement | Enamine formation/C–C bond formation rate-limiting | kpol ≈ 10–300 s⁻¹ (chemistry step partly rate-limiting) |
Test yourself
Try to answer each question before opening it.
Which reagents and conditions are typical for the Serine protease?
Peptide substrate, chymotrypsin/trypsin-like protease (Ser195, His57, Asp102), H₂O. Conditions: Aqueous, pH ≈ 7–8, 37 °C.
Which bonds are formed and which are broken?
Formed: Ser195 acyl-enzyme ester; free carboxylic acid; free amine. Broken: Peptide C–N; Ser195 O–H.
What is the stereochemical outcome?
Oxyanion hole (Gly193, Ser195 N–H) stabilises tetrahedral O⁻
What controls the rate or the selectivity?
Acylation and deacylation both contribute; ~10¹⁰-fold rate enhancement
Study guides that help with this reaction
- How to learn reaction mechanisms (without memorising everything)
- Curved-arrow notation: rules and the mistakes students make