Abstract
Rat trypsin II has been converted to a protease with chymotrypsin-like substrate specificity
[Hedstrom, L., et al. (1994) Biochemistry (preceding paper in this issue)]. The key alteration in this
conversion is the exchange of two surface loops for the analogous loops of chymotrypsin. k\mct/K{ for the
inactivation of chymotrypsin, trypsin, a trypsin mutant with poor activity (D189S), and the chymotrypsinlike mutants Tr—Ch [Sl+Ll+L2] and Tr—Ch [Sl+Ll+L2+Y172 W] by Suc-Ala-Ala-Pro-Phe-chloromethylketone correlates with kai/Km for hydrolysis of Sue-Ala-Ala-Pro-Phe-AMC. kmact’s for the inactivation
of Tr—Ch [Sl+Ll +L2] and T r—Ch[S 1+L1+L2+Y17 2 W] are comparable to that of chymotrypsin, while
K's were much higher. K¡ for the inhibition of these enzymes by the transition-state analog MeOSucAla-Ala-Pro-boro-Phe also correlates with kCit/Km for hydrolysis of Sue-Ala-Ala-Pro-Phe-AMC. These
results suggest that the surface loops stabilize the transition state for hydrolysis of chymotrypsin substrates
by improving the orientation of bound substrates relative to the catalytic residues. Lastly, trypsin and
chymotrypsin have comparable affinities for proflavin, while the K¿ for the Tr—Ch [S1+L1+L2+Y172W]-
proflavin complex is 10-fold higher. No proflavin binding could be observed for either D189S or Tr—Ch-
[S1+L1+L2], which suggests that the SI binding pockets of these two mutant enzymes are deformed. This
work confirms that enzyme specificity is expressed in the chemical steps of the reaction rather than in
substrate binding.