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The 26,000-Mr DD-peptidase of Streptomyces K15 binds one equivalent of thiol reagents as 5,5′-dithiobis-(2-nitrobenzoate) or p-chloromercuribenzoate (pCMB). Derivatization of the DD-peptidase by pCMB decreases the efficacy of the initial binding of the ester carbonyl donor Ac2-L-Lys-D-Ala-D-lactate to the enzyme (K), the rate of enzyme acylation by the donor (K+2) and the rate of enzyme deacylation (k+3). However, the value of the k+2/k+3 ratio, and therefore the percentage of total enzyme which, at saturating concentrations of the donor, is present as acyl-enzyme at the steady state of the reaction, are not modified. The enzyme's binding sites for pCMB and benzylpenicillin are not mutually exclusive. But, when compared with the native enzyme, the pCMB-derivatized enzyme undergoes acylation by benzylpenicillin with a decreased second-order-rate constant (k+2/K) value and gives rise to a penicilloyl adduct of increased stability. Since the acyl-enzyme mechanism is not annihilated by pCMB derivatization, it is proposed that basically, and like all the other DD-peptidases/penicillin-binding proteins so far characterized, the Streptomyces K15 DD-peptidase is an active-site-serine enzyme.

The values of the kinetic parameters that govern the interactions between the Streptomyces K15 DD-peptidase and beta-lactam compounds were determined by measuring the inactivating effect that these compounds exert on the transpeptidase activity of the enzyme and, in the case of [14C]benzylpenicillin and [14C]cefoxitin, by measuring the amounts of acyl-enzyme formed during the reaction. K15 DD-peptidase binds benzylpenicillin or cefoxitin at a molar ratio of 1:1. Benzylpenicilloate is the major product released during breakdown of the acyl-enzyme formed with benzylpenicillin. Benzylpenicillin is not a better acylating agent than the amide Ac2-L-Lys-D-Ala-D-Ala and ester Ac2-L-Lys-D-Ala-D-lactatecarbonyl-donor substrates. beta-Lactam compounds possessing a methoxy group on the alpha-face of the molecule show high inactivating potency.