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-GLYn-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-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 decrease 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 proflavine.
D000166:Acridines / Q000494:pharmacology*; D001120:Arginine / Q000031:analogs & derivatives / Q000378:metabolism; D001391:Azo Compounds / Q000378:metabolism*; D001565:Benzoates / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D004151:Dipeptides / Q000378:metabolism; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D010206:Papain / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D011370:Proflavine / Q000494:pharmacology*; D013379:Substrate Specificity
D000166:Acridines; D001391:Azo Compounds; D001565:Benzoates; D004151:Dipeptides; D010455:Peptides; D001120:Arginine; D011370:Proflavine; D010206:Papain