Kinetics of β-lactam interactions with penicillin-susceptible and -resistant penicillin-binding protein 2x proteins from Streptococcus pneumoniae -: Involvement of acylation and deacylation in β-lactam resistance

Kinetic interactions of beta -lactam antibiotics such as penicillin-G and cefotaxime with normal, penicillin-susceptible PBP2x from Streptococcus pneumoniae and a penicillin-resistant PBP2x (PBP2x(R)) from a resistant clinical isolate (CS109) of the bacterium have been extensively characterized using electrospray mass spectrometry coupled with a fast reaction (quench flow) technique. Kinetic evidence for a two-step acylation of PBP2x by penicillin-G has been demonstrated, and the dissociation constant, K-d of 0.9 mM, and the acylation rate constant, k(2) of 180 s(-1), have been determined for the first time. The millimolar range K-d implies that the beta -lactam fits to the active site pocket of the penicillin-sensitive PEP rather poorly, whereas the extremely fast k(2) value indicates that this step contributes most of the binding affinity of the beta -lactam. The values of K-d (4 mM) and k(2) (0.56 s(-1)) were also determined for PBP2x(R). The combined value of k(2)/K-d, known as overall binding efficiency, for PBP2xR (137 M-1 s(-1)) was over 1000-fold slower than that for PBP2x (200,000 M-1 s(-1)), indicating that a major part is played by the acylation steps in penicillin resistance. Most of the decreased binding efficiency of PBP2xR comes from the decreased (similar to 300-fold) k(2) Kinetic studies of cefotaxime acylation of the two PBP2x proteins confirmed all of the above findings. Deacylation rate constants (k(3)) for the third step of the interact-ions were determined to be 8 x 10(-6) s(-1) for penicilloyl-PBP2x and 5.7 x 10(-4) s(-1) for penicilloyl-PBP2x(R), corresponding to over 70-fold increase of the deacylation rate for the resistant PBP2xR. Similarly, over 80-fold enhancement of the deacylation rate was found for cefotaxime-PBP2x(R) complex (k(3) = 3 x 10(-4) s(-1)) as compared with that of cefotaxime-PBP2x complex (3.5 x 10-6 s(-1)). This is the first time that such a significant increase of k(3) values was found for a beta -lactsm-resistant penicillin-binding protein. These data indicate that the deacylation step also plays a role, which is much more important than previously thought, in PBP2x(R) resistance to beta -lactams.