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Science 6 March 1992:
Vol. 255. no. 5049, pp. 1249 - 1253
DOI: 10.1126/science.1546324
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Articles

Science, Vol 255, Issue 5049, 1249-1253
Copyright © 1992 by American Association for the Advancement of Science

articles

Converting trypsin to chymotrypsin: the role of surface loops

L Hedstrom, L Szilagyi, and WJ Rutter

Hormone Research Institute, University of California, San Francisco 94143-0534.

Trypsin (Tr) and chymotrypsin (Ch) have similar tertiary structures, yet Tr cleaves peptides at arginine and lysine residues and Ch prefers large hydrophobic residues. Although replacement of the S1 binding site of Tr with the analogous residues of Ch is sufficient to transfer Ch specificity for ester hydrolysis, specificity for amide hydrolysis is not transferred. Trypsin is converted to a Ch-like protease when the binding pocket alterations are further modified by exchange of the Ch surface loops 185 through 188 and 221 through 225 for the analogous Tr loops. These loops are not structural components of either the S1 binding site or the extended substrate binding sites. This mutant enzyme is equivalent to Ch in its catalytic rate, but its substrate binding is impaired. Like Ch, this mutant utilizes extended substrate binding to accelerate catalysis, and substrate discrimination occurs during the acylation step rather than in substrate binding.


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