Dissecting the catalytic triad of a serine protease.
Level 5 - mechanism / opinion, no new human data
Level 5 by design analogy (in vitro bench research and site-directed mutagenesis)
PubMed 3282170 · doi:10.1038/332564a0
What was done
Investigators evaluated the individual and collective catalytic contributions of the Ser-His-Asp catalytic triad in Bacillus amyloliquefaciens subtilisin. Using site-directed mutagenesis on the cloned gene, residues within the catalytic triad were individually or multiply replaced with alanine to minimize steric disruption and avoid introducing novel side-chain interactions. Kinetic parameters, specifically turnover number and the Michaelis constant, were measured for the mutant enzymes.
What was found
Mutations within the catalytic triad substantially reduced the turnover number while producing only minor effects on the Michaelis constant. Kinetic analyses of multiple mutants demonstrated that the triad residues interact synergistically to accelerate amide bond hydrolysis by a factor of approximately 2 x 10(6), contributing to the total wild-type rate enhancement of at least 10(9) to 10(10) times non-enzymatic hydrolysis.
Why it matters
This work provides direct experimental quantification of synergistic cooperation among residues in the classic serine protease catalytic triad. It clarifies how coordinated active-site side chains achieve massive transition-state stabilization during peptide bond cleavage.
Limits
Findings are derived from in vitro kinetic assays on a single bacterial protease (Bacillus amyloliquefaciens subtilisin). Specific kinetic values for individual alanine mutants and sample replicates are not reported in the abstract.
Cited by
- supports Replacing the active site serine's hydroxyl oxygen with a hydrogen turns the residue into alanine and renders a serine protease completely functionless without altering its 3D structure.