A NEW KINETIC MECHANISM FOR THE CONCOMITANT HYDROLYSIS AND TRANSFER-REACTIONS CATALYZED BY BACTERIAL DD-PEPTIDASES

In the presence of an adequate nucleophilic acceptor substrate (A) and ester and thiolester donor substrates (S), the Streptomyces R61 solub le DD-peptidase catalyzes both hydrolysis and acyl group transfer reac tions. Simple bisubstrate models do not explain the variations of the transfer to hydrolysis ratios with the donor and acceptor concentratio ns. A new kinetic mechanism for the concomitant hydrolysis and transfe r reactions is proposed which involves an acceptor and a second, nonpr oductive donor substrate binding site. In this model, the acceptor ess entially binds to the acyl-enzyme, and the second donor molecule only binds to the ternary ESA complex. Hydrolysis can then proceed from th e quaternary ESAS complex. The values of all of the parameters involv ed in the reaction of a thiolester substrate with D-alanine as the acc eptor substrate were determined at 15 and 37-degrees-C. The results ob tained with a protein modified by site-directed mutagenesis, and with which the transpeptidation reaction appeared to be specifically impede d, are discussed on the basis of the new kinetic mechanism. The data o btained with the soluble form of the high molecular weight penicillin binding protein 2X from Streptococcus pneumoniae are also in agreement with this model.