Reaction of soluble penicillin-binding protein 2a of methicillin-resistant Staphylococcus aureus with β-lactams and acyclic substrates: Kinetics in homogeneous solution

Abstract

The kinetics of reaction of solubilized penicillin-binding protein 2a (sPBP2a) of methicillin-resistant Staphylococcus aureus with a variety of β-lactams and acyclic species was studied in homogeneous aqueous solution at 37°C in 25mM Hepes buffer, pH 7.0, containing 1 M NaCl. Under these conditions, but not at lower salt concentrations, protein precipitation did not occur either during or after the reaction. The reactions of β-lactams in general could be monitored by competition with a chromophoric β-lactam, nitrocefin, or directly in certain cases by protein fluorescence. Rate constants for reaction of a wide variety of β-lactams are reported. The interactions are characterized by a slow second-order acylation reaction followed by a slower deacylation. For example, the rate constants for benzylpenicillin were 12 M-1.s-1 and 3 x 10-5 s-1 respectively. The acylation is slow in comparison with those of normal non-resistant highmolecular-mass penicillin-binding proteins. sPBP2a also seemed to catalyse the slow hydrolysis of a variety of acyclic depsipeptides but not that of a D-Ala-D-Ala peptide. The reactions with certain depsipeptides also led to protein precipitation. These reactions were, however, not affected by prior blockage of the β-lactam-binding site by benzylpenicillin and thus might take place elsewhere on the enzyme. Two classes of potential transitionstate analogue inhibitors, phosphonate monoesters and boronates, seemed to have little effect on the rate of reaction of sPBP2a with nitrocefin and therefore seem to have little affinity for the β-lactam-binding/D,D-peptidase site.