Abstract
Serine proteases are present in virtually all organisms and function both inside and outside the cell1; they exist as two families, the 'trypsin-like' and the 'subtilisin-like', that have independently evolved a similar catalytic device2 characterized by the Ser, His, Asp triad, an oxyanion binding site, and possibly other determinants that stabilize the transition state (Fig. l) 2–4. For Bacillus amyloliquefaciens subtilisin, these functional elements impart a total rate enhancement of at least 109 to 1010 times the non-enzymatic hydrolysis of amide bonds. We have examined the catalytic importance and interplay between residues within the catalytic triad by individual or multiple replacement with alanine(s), using site-directed mutagenesis5,6 of the cloned B. amyloliquefaciens subtilisin gene7. Alanine substitutions were chosen to minimize unfavourable steric contacts and to avoid imposing new charge interactions or hydrogen bonds from the substituted side chains. In contrast to the effect of mutations in residues involved in substrate binding8–10, the mutations in the catalytic triad greatly reduce the turnover number and cause only minor effects on the Michaelis constant. Kinetic analyses of the multiple mutants demonstrate that the residues within the triad interact synergistically to accelerate amide bond hydrolysis by a factor of ∼2×l06.
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Carter, P., Wells, J. Dissecting the catalytic triad of a serine protease. Nature 332, 564–568 (1988). https://doi.org/10.1038/332564a0
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DOI: https://doi.org/10.1038/332564a0
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