Chemical modifications of the active site of Streptomyces R61 DD-carboxypeptidase.

The DD-carboxypeptidase of Streptomyces R61 is an exocellular enzyme related to the bacterial peptidoglycan cross-linking enzymes, and, like them, is inhibited by penicillin. The active-site reagents methanesulfonyl fluoride and diisopropylfluorophosphate inhibit catalytic activity and binding of penicillin G indicating the involvement of a serine residue in both processes. For methanesulfonyl fluoride the second-order rate constant (0.7 M-1 min-1) is comparable to that of classical serine proteases. For diisopropylfluorophosphate, which binds to the enzyme stoichiometrically, the second-order rate constant (1.5 M-1 min-1) is at least two orders of magnitude smaller. The arginine-specific reagents methylglyoxal, 2,3-butanedione and phenylglyoxal inactive DD-carboxypeptidase in borate buffer with second-order rate constants of 70, 70 and 120 M-1 min-1, respectively. Inactivation correlates with stoichiometric binding to the enzyme. Peptidase and esterase activities are similarly affected, suggesting that substrate binding in both cases requires an arginine-carboxyl group interaction. Penicillin binding is also inhibited, but the degree of inhibition depends on the alpha-dicarbonyl side chain. Binding of alpha-dicarbonyls to DD-carboxypeptidase facilitates subsequent binding of diisopropylfluorophosphate suggesting that interaction of these compounds with the active site might induce a conformational change on the enzyme making the serine residue more accessible to the modifying reagent.