SOLUBLE PENICILLIN-BINDING PROTEIN 2A - BETA-LACTAM BINDING AND INHIBITION BY NON-BETA-LACTAMS USING A 96-WELL FORMAT

High level methicillin resistance in Staphylococcus aureus is dependen t upon the acquisition of the mecA gene encoding penicillin-binding pr otein 2a (PBP2a). PBP2a is a member of a family of peptidoglycan biosy nthetic enzymes involved in assembly of the cell wall in bacteria and is poorly inactivated by beta-lactam antibiotics, We describe a 96-wel l-filter binding assay using recombinant, soluble PBP2a which allows f or kinetic measurement of penicillin binding. The deacylation rate con stant for the PBP2a-penicillin G covalent complex was found to be 5.7 +/- 1.0 x 10(-5) s(-1) at 30 degrees C (half-life of similar to 200 mi n). For the PBP2a acylation reaction, the value of K-m (penicillin G) = 0.5 +/- 0.1 mM and k(cat) = 1 x 10(-3) s(-1), which yields a second- order rate constant (k(cat)/K-m) for inactivation of 2.0 M-1 s(-1) Usi ng this assay, several non-beta-lactam inhibitors including Cibacron b lue have been found which exhibit IC50 values between 10 and 30 mu M. The binding affinities of several carbapenems and beta-lactams correla ted well between the filter binding assay described in this report and an electrophoretic assay for PBP2a using membranes prepared from meth icillin-resistant S. aureus. (C) 1998 Academic Press.