Studies on papain catalysis with substrates containing the Trans-p-phenylazobenzoyl group as a probe for hydrophobic interaction

The susceptible bonds of substrates of the type PABz-Gly-Arg-R (R=OCH3 n=0,1,2; R=OH, n=1,2) to papain [EC 3.4.22.2] have been identified. PABz-Arg-OMe was found to be approximately 30 times more reactive than benzoylarginine ethyl ester, due to a Km(app) value 25 times smaller. The finding that PABz-Gly2 was specifically hydrolyzed at the ester bond with a second-order rate constant comparable with that of PABz-Gly2-Arg-OMe indicated that the S4site of the enzyme is capable of accommodating a large hydrophobic group. Other substrates were cleaved at the peptide bond one residue removed from the PABz group, in accord with the well-known fact that the S2 subsite interacts preferentially with a hydrophobic P2 residue. A negative charge on the substrate at the P2′ or P3′ position greatly decreases the acylation rate and also the noncovalent binding affinity. The hydrolysis of PABz-Arg-OMe and PABz-Gly2-OMe was little affected by the acridine dye proflavine