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

Citation
Wp. Lu et al., Kinetics of beta-lactam interactions with penicillin-susceptible and -resistant penicillin-binding protein 2x proteins from Streptococcus pneumoniae - Involvement of acylation and deacylation in beta-lactam resistance, J BIOL CHEM, 276(34), 2001, pp. 31494-31501
Citations number
37
Categorie Soggetti
Biochemistry & Biophysics
Journal title
JOURNAL OF BIOLOGICAL CHEMISTRY
ISSN journal
00219258 → ACNP
Volume
276
Issue
34
Year of publication
2001
Pages
31494 - 31501
Database
ISI
SICI code
0021-9258(20010824)276:34<31494:KOBIWP>2.0.ZU;2-L
Abstract
Kinetic interactions of beta -lactam antibiotics such as penicillin-G and c efotaxime with normal, penicillin-susceptible PBP2x from Streptococcus pneu moniae and a penicillin-resistant PBP2x (PBP2x(R)) from a resistant clinica l isolate (CS109) of the bacterium have been extensively characterized usin g electrospray mass spectrometry coupled with a fast reaction (quench flow) technique. Kinetic evidence for a two-step acylation of PBP2x by penicilli n-G has been demonstrated, and the dissociation constant, K-d of 0.9 mM, an d 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, w hereas the extremely fast k(2) value indicates that this step contributes m ost of the binding affinity of the beta -lactam. The values of K-d (4 mM) a nd 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)), indica ting that a major part is played by the acylation steps in penicillin resis tance. Most of the decreased binding efficiency of PBP2xR comes from the de creased (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 determin ed 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 deacyla tion 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) va lues was found for a beta -lactsm-resistant penicillin-binding protein. The se data indicate that the deacylation step also plays a role, which is much more important than previously thought, in PBP2x(R) resistance to beta -la ctams.