Characterization of the S 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 / K , for tripeptide substrate hydrolysis was observed to increase in the order Glu < Gly < Arg < Val < Tyr. Molecular modeling studies of substrates containing a P 3 Glu, Arg, or Tyr covalently bound as the tetrahedral intermediate to the enzyme suggest that the specificity for a P 3 Tyr is because of a favorable aromatic-aromatic interaction with Tyr on the enzyme as well as a possible H bond between the P 3 Tyr hydroxyl and the side chain carboxyl of Asp .