PURIFICATION, PROPERTIES, AND KINETICS OF ENZYMATIC ACYLATION WITH BETA-LACTAMS OF SOLUBLE PENICILLIN-BINDING PROTEIN 2A - A MAJOR FACTOR IN METHICILLIN-RESISTANT STAPHYLOCOCCUS-AUREUS

Intrinsic resistance toward beta-lactams in methicillin-resistant Stap hylococcus aureus strains, a major source of nosocomial infections, is believed to be caused mainly by penicillin-binding protein 2a (PBP2a) . This protein resembles other penicillin-binding proteins that are in volved in bacterial cell wall biosynthesis and are the targets of act ive site acylation by beta-lactam antibiotics. PBP2a, however, presuma bly remains active at thera peutic concentrations of beta-lactams. In this paper, we describe a three-step purification of a soluble form of PBP2a (PBP2a') to apparent homogeneity using anion-and cation-exchang e, and dye ligand affinity chro matographies. Purified PBP2a' was a 74 -kDa monomeric protein that appeared to be folded. The protein was eva luated for its enzymatic acylation with beta-lactams initially by fluo rescence quenching and then kinetically by radioactive labeling. Using a modified I-125-labeled penicillin V-acylation assay, the apparent K -m of PBP2a' for penicillin V was 1.2 mM. Three other beta-lactams, ea ch of which exhibited significant fluorescence quenching, acted as str ong competitive inhibitors of penicillin V with apparent K-i values of 123.4, 36.1, and 12.4 mu M, respectively. By a new beta-lactam preinc ubation analysis, these compounds could function as substrates with si milar K-m values. Also, the acylation rates of different beta-lactams could be readily ascertained. The enzymatic acylation data substantiat e the major causative role of PBP2a in the bacterial resistance. The q uantitative radioactive acylation assays are potentially useful in scr eening for a potent inhibitor of the enzyme.