Converting Trypsin to Chymotrypsin: Residue 172 Is a Substrate Specificity Determinant

Abstract

Trypsin and chymotrypsin have very similar tertiary structures, yet very different substrate specificities. Recent site-directed mutagenesis studies have shown that mutation of the residues of the substrate binding pocket of trypsin to the analogous residues of chymotrypsin does not convert trypsin into a protease with chymotrypsin-like specificity. However, chymotrypsin-like substrate specificity is attained when two surface loops are changed to the analogous residues of chymotrypsin, in conjunction with the changes in the S1 binding site [Hedstrom, L.,Szilagyi, L., & Rutter, W. J. (1992) Science 255,1249–1253). This mutant enzyme, Tr→Ch[S1+L1+L2], is improved to a protease with 2–15% of the activity of chymotrypsin by the mutation of Tyr172 to Trp. Residue 172 interacts synergistically with the residues of the substrate binding pocket and the loops to determine substrate specificity. Further, these trypsin mutants demonstrate that substrate specificity is determined by the rate of catalytic processing rather than by substrate binding. © 1994, American Chemical Society. All rights reserved.