CONVERTING TRYPSIN TO CHYMOTRYPSIN - RESIDUE-172 IS A SUBSTRATE-SPECIFICITY DETERMINANT

Trypsin and chymotrypsin have very similar tertiary structures, yet ve ry different substrate specificities. Recent site-directed mutagenesis studies have shown that mutation of the residues of the substrate bin ding 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 tw o surface loops are changed to the analogous residues of chymotrypsin, in conjunction with the changes in the S1 binding site [Hedstrom, L., Szilagyi, L., and 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. Res idue 172 interacts synergistically with the residues of the substrate binding pocket and the loops to determine substrate specificity. Furth er, these trypsin mutants demonstrate that substrate specificity is de termined by the rate of catalytic processing rather than by substrate binding.