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 SI binding site [Hedstrom, L., Szilagyi, L., & Rutter, W. J. (1992) Science 255,1249-1253).
This mutant enzyme, Tr-*-Ch[Sl+Ll+L2], is improved to a protease with 2-15% of the activity of
chymotrypsin by the mutation of Tyrl72 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.