Abstract
The conversion of trypsin into a protease with chymotrypsin-like activity and specificity required substitution of fifteen residues in the S1 site and two surface loops with their chymotrypsin counterparts [Hedstrom,L., Szilagyi,L. and Rutter,W.J. (1992) Science, 255, 1249-1253]. These residues may define a set of general structural determinants of specificity in the trypsin family. In order to test this hypothesis, we have attempted to convert trypsin into a protease with specificity for substrates containing small aliphatic residues by replacing the S1 site and these surface loops with the analogous residues of elastase. Five elastase-like mutant enzymes were constructed with various combinations of these substitutions. Four mutant enzymes catalyze the hydrolysis of MeOSuc-Ala-Ala-Pro-Ala-SBzl more efficiently than the hydrolysis of Suc-Ala-Ala-Pro-Phe-SBzl. This observation indicates that the mutant enzymes have elastase-like esterase specificity. The best mutant, Tr-->E1-2, is a more specific esterase than elastase: the ratio of the values of kcat/Km for MeOSuc-Ala-Ala-Pro-Ala-SBzl and Suc-Ala-Ala-Pro-Phe-SBzl is greater than 160 for Tr-->E1-2 and 50 for elastase. However, the esterase activity of Tr-->E1-2 is 300-fold less than elastase; in addition, Tr-->E1-2 has no measurable amidase activity. Thus these substitutions do not construct a protease with elastase-like activity. These experiments indicate that a unique structural solution is required for each different specificity. Previous work suggested that instability of the S1 site is a major barrier to redesigning the specificity of trypsin. This view is corroborated by preliminary structural studies of Tr-->E1-2. One dimensional 1H NMR spectrum of Tr-->E1-2 suggests that the S1 site and the two surface loops of this mutant trypsin may be disordered.