Penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus:: Kinetic characterization of its interactions with β-lactams using electrospray mass spectrometry

Penicillin-binding protein 2a (PBP2a) is the primary beta-lactam resistance determinant of methicillin-resistant Staphylococcus aureus (MRSA). MecA, the gene coding for PBP2a, was cloned with the membrane-anchoring region at the N-terminus deleted. The truncated protein (PBP2a*) was overexpressed in Escherichia coli mostly in the soluble form accounting for similar to 25% of soluble cell protein and was purified to homogeneity. The purified protein was shown to covalently bind beta-lactams in an 1:1 ratio as determined by electrospray mass spectrometry, A novel method based on HPLC-elctrospray mass spectrometry has been developed to quantitatively determine the formation of the covalent adducts or acyl-PBP2a* complexes. By using this method, combined with kinetic techniques including quench flow, we have extensively characterized the interactions between PBP2a* and three beta-lactams and determined related kinetic parameters for the first time. The apparent first-order rate constants (k(a)) of PBP2a* acylation by benzylpenicillin showed a hyperbolic dependence on the concentration of benzylpenicillin. This is consistent with the mechanism that the binding of the penicillin to PBP2a* consists of reversible formation of a Michaelis complex followed by formation of the penicilloyl-PBP2a* adduct, and allowed the determination of the individual kinetic parameters for these two steps, the dissociation constant K-d of 13.3 mM and the first-order rate constant k(2) of 0.22 s(-1). From these values, the second-order rate constant k(2)/K-d, the value reflecting the overall binding efficiency of a beta-lactam, of 16.5 M-1 s(-1) was obtained. The fairly high K-d value indicates that benzylpenicillin fits rather poorly into the protein active site. Similar studies on the interaction between PBP2a* and methicillin revealed k(2) of 0.0083 s(-1) and K-d Of 16.9 mM, resulting in an even smaller k(2)/K-d value of 0.49 M-1 s(-1). The rate constants k(3) for deacylation of the acyl-PBP2a* complexes, the third step in the interactions, were measured to be (1.5 x 10(-5) s(-1). These results indicate that the resistance of PBP2a to penicillin inactivation is mainly due to the extremely low penicillin acylating rate in addition to the low association affinity, but not to a fast rate of deacylation. Acylation of PBP2a* by a high-affinity cephalosporin, Compound 1, also followed a saturation curve of k(a) versus the compound concentration, from which k(2) = 0.39 s(-1), K-d = 0.22 mM, and k(2)/K-d = 1750 M-1 s(-1) were obtained. The 100-fold increase in the k(2)/K-d value as compared with that of benzylpenicillin is mostly attributable to the decreased (60-fold) K-d, indicating that the cephalosporin fits much better to the binding pocket of the protein.