Studies on Papain Catalysis with Substrates Containing the Trans-p-Phenylazobenzoyl Group as a Probe for Hydrophobic Interaction

抄録

The susceptible bonds of substrates of the type PABz-Gly_n-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 K_m(app) value 25 times smaller. The finding that PABz-Gly₂-Arg-OMe was specifically hydrolyzed at the ester bond with a second-order rate constant comparable with that of PABz-Arg-OMe indicated that the S₄ subsite 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-Arg-OMe was little affected by the acridine dye profiavine.