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 the primary enzyme responsible for the β‐lactam resistance in methicillin‐resistant Staphylococcus aureus (MRSA). Inhibition of a PBP such as PBP2a by β‐lactams is due to covalent modification of an active site serine residue. Based on the sequence alignment with well studied β‐lactamases, DD‐carboxypeptidases and other high molecular mass PBPs, the serine of a tetrad S403XXK in PBP2a was tentatively identified as the penicillin‐binding site. However, direct evidence for the involvement of serine403 has not been reported. In this study, a method which combines liquid chromatography/electrospray mass spectrometry (LC/MS) and nano‐electrospray MS for the identification of the active site serine in PBP2a is described. The covalent binding of the β‐lactams was carried out in vitro with the recombinant PBP2a. Peptide mapping of the cyanogen bromide fragments from penicilloyl‐PBP2a, using microbore LC/MS, provided a rapid identification of the modified peptide with a 334 Da mass increase. The acylated peptide was isolated and further 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. © 1998 John Wiley & Sons, Ltd.