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 beta-lactams and acyclic species was studied in homogeneous aqueous solution at 37 degreesC in 25 mM Hepes buffer, pH7.0, 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 beta-lactams in general could be monitored by competition with a chromophoric beta-lactam, nitrocefin, or directly in certain cases by protein fluorescence. Rate constants for reaction of a wide variety of beta-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 3x10(-5) s-1 respectively. The acylation is slow in comparison with those of normal non-resistant high-molecular-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 beta-lactam-binding site by benzylpenicillin and thus might take place elsewhere on the enzyme. Two classes of potential transition- state 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 beta-lactam-binding/D,D-peptidase site.

Co-opting the Cell Wall in Fighting Methicillin-Resistant Staphylococcus aureus: Potent Inhibition of PBP 2a by Two Anti-MRSA β-Lactam Antibiotics

“…Additionally, we were not able to detect any deacylation of the CPT or ME acyl-enzyme species, while monitoring for four days. Previously it was reported that upon acylation by antibiotics PBP 2a precipitates, 13 or alternatively an oligomerization of the acylated protein could explain the sheltering of the acyl-enzyme species to impart longevity. Neither of these two possibilities is applicable in the cases of CPT or ME binding to PBP 2a, as detailed in Supporting Information.…”

“…In some instances, the E-I complex undergoes deacylation, but the rate of such deacylation is sufficiently slow that the bacterium is deprived of the enzyme function and dies. 7,13 (1)…”

The Basis for Resistance to β-Lactam Antibiotics by Penicillin-binding Protein 2a of Methicillin-resistant Staphylococcus aureus

“…Nitrocefin (120 M) was used as the reporter molecule to determine the apparent first-order rate constants for acylation by other nonchromogenic (or poorly chromogenic) ␤-lactams at varying concentrations in competition experiments (18).…”

Interaction energies between β‐lactam antibiotics and E. coli penicillin‐binding protein 5 by reversible thermal denaturation

“…These studies provide the first quantitative measurement of the noncovalent interaction energies of a PBP with covalently bound ␤-lactam antibiotics. Intriguingly, these noncovalent interaction energies do not correlate with the second-order rate constants for formation of the acyl-enzyme complex (k 2 /KЈ), which are considered the best indicators of the inhibitory potency of ␤-lactam antibiotics with PBPs (Frere et al 1975;Frere et al 1992;Jamin et al 1993;Graves-Woodward and Pratt 1998;Lu et al 1999).…”