Soluble Penicillin-Binding Protein 2a: β-Lactam Binding and Inhibition by Non-β-Lactams Using a 96-Well Format

High level methicillin resistance inStaphylococcus aureusis dependent upon the acquisition of themecAgene encoding penicillin-binding protein 2a (PBP2a). PBP2a is a member of a family of peptidoglycan biosynthetic enzymes involved in assembly of the cell wall in bacteria and is poorly inactivated by β-lactam antibiotics. We describe a 96-well-filter binding assay using recombinant, soluble PBP2a which allows for kinetic measurement of penicillin binding. The deacylation rate constant for the PBP2a–penicillin G covalent complex was found to be 5.7 ± 1.0 × 10−5s−1at 30°C (half-life of ∼ 200 min). For the PBP2a acylation reaction, the value ofKm(penicillin G) = 0.5 ± 0.1 mM andkcat= 1 × 10−3s−1, which yields a second-order rate constant (kcat/Km) for inactivation of 2.0 M−1s−1. Using this assay, several non-β-lactam inhibitors including Cibacron blue have been found which exhibit IC50values between 10 and 30 μM. The binding affinities of several carbapenems and β-lactams correlated well between the filter binding assay described in this report and an electrophoretic assay for PBP2a using membranes prepared from methicillin-resistantS. aureus.