CHARACTERIZATION OF THE S-3 SUBSITE SPECIFICITY OF CATHEPSIN-B

Five synthetic substrates containing different amino acid residues at the P-3 position (acetyl-X-Arg-Arg-AMC, where X is Gly, Glu, Arg, Val, and Tyr and where AMC represents 7-amido-4-methylcoumarin) were used to investigate the S-3 subsite specificity of cathepsin B. At pH 6.0, the specificity constant, k(cat)/K-m, for tripeptide substrate hydroly sis was observed to increase in the order Glu < Gly < Arg < Val < Tyr. Molecular modeling studies of substrates containing a P-3 Glu, Arg, o r Tyr covalently bound as the tetrahedral intermediate to the enzyme s uggest that the specificity for a P-3 Tyr is because of a favorable ar omatic-aromatic interaction with Tyr(75) on the enzyme as well as a po ssible H bond between the P-3 Tyr hydroxyl and the side chain carboxyl of Asp(69).