IDENTIFICATION OF THE ACTIVE-SITE SERINE OF PENICILLIN-BINDING PROTEIN 2A FROM METHICILLIN-RESISTANT STAPHYLOCOCCUS-AUREUS BY ELECTROSPRAY MASS-SPECTROMETRY

Penicillin-binding protein 2a (PBP2a), a high molecular mass PBP, is t he primary enzyme responsible for the beta-lactam resistance in methic illin-resistant Staphylococcus aureus (MRSA). Inhibition of a PBP such as PBP2a by beta-lactams is due to covalent modification of an active site serine residue. Based on the sequence alignment with well studie d beta-lactamases, DD-carboxypeptidases and other high molecular mass PBPs, the serine of a tetrad S403XXK in PBP2a was tentatively identifi ed as the penicillin-binding site. However, direct evidence for the in volvement of serine403 has not been reported. In this study, a method which combines liquid chromatography/electrospray mass spectrometry (L C/MS) and nano-electrospray MS for the identification of the active si te serine in PBP2a is described. The covalent binding of the beta-lact ams was carried out in vitro with the recombinant PBP2a. Peptide mappi ng of the cyanogen bromide fragments from penicilloyl-PBP2a, using mic robore LC/MS, provided a rapid identification of the modified peptide with a 334 Ha mass increase. The acylated peptide was isolated and fur ther digested with trypsin. Nano-electrospray MS/MS sequencing of the acylated peptide in the tryptic digest showed that the penicillin was indeed attached to serine403. (C) 1998 John Wiley & Sons, Ltd.