Use of N-benzoyl-L-tyrosine thiobenzyl ester as a protease substrate. Hydrolysis by alpha-chymotrypsin and subtilisin BPN.

In the course of searching for specific chromogenic substrates which might be useful in screening for protease-deficient mutants of Bacillus subtilis, we have developed a method for the synthesis of N-benzoyl-L-tyrosine thiobenzyl ester (BzTyrSBzl) in good yield. Spontaneous base hydrolysis of this thiol ester is low, but several serine proteases hydrolyze it readily. Spectrophotometric measurement of the hydrolysis of the ester in the presence of 5,5'-dithiobis(2-nitrobenzoic acid) provides a continuous assay for chymotrypsin as sensitive as any assay reported in the literature. Serine proteases which hydrolyze this substrate may be detected in polyacrylamide disc gels by incubation in the presence of nitro blue tetrazolium. Apparent Km values of 0.02 and 7 mM and kcat values of 37 S-1 and 126 S-1 were observed for the hydrolysis of BzTyrSBzl by alpha-chymotrypsin and subtilisin BPN', respectively. Additionally, 5 mM indole was observed to behave as a strict competitive inhibitor of the alpha-chymotrypsin-catalyzed hydrolysis of BzTyrSBzl but was observed to increase the maximal rate of hydrolysis of p-nitrophenyl acetate by alpha-chymotrypsin by 30%, as previously described. These data, the published data of other workers, and results from studies with molecular models of trypsin and subtilisin BPN' are used as the basis for describing more fully a secondary hydrophobic binding pocket on alpha-chymotrypsin. The pocket is immediately adjacent to the active site serine and is tentatively suggested to be composed of 4 aliphatic side chain residues and 2 glycine residues.