Hydrogen bonding and protein perturbation in β-lactam acyl-enzymes of Streptococcus pneumoniae penicillin-binding protein PBP2x

A soluble form of Streptococcus pneumoniae PBP2x, a molecular target of penicillin and cephalosporin antibiotics, has been expressed and purified. IR difference spectra of PBP2x acylated with benzylpenicillin, cloxacillin, cephalothin and ceftriaxone have been measured. The difference: spectra show two main features. The ester carbonyl vibration of the acyl-enzyme is ascribed to a small band between 1710 and 1720 cm(-1), whereas a much larger band at approx. 1640 cm(-1) is ascribed to a perturbation in the structure of the enzyme, which occurs on acylation. The protein perturbation has been interpreted as occurring in P-sheet. The acyl-enzyme formed with benzylpenicillin shows the lowest ester carbonyl vibration frequency, which is interpreted to mean that the carbonyl oxygen is the most strongly hydrogen-bended in the oxyanion hole of the antibiotics studied. The semi-synthetic penicillin cloxacillin is apparently less well organized in the active site and shows two partially overlapping ester carbonyl bands. The penicillin acyl-enzyme has been shown to deacylate more slowly than that formed with cloxacillin. This demonstrates that the natural benzylpenicillin forms a more optimized and better-bonded acyl-enzyme and that this in turn leads to the stabilization of the acyl-enzyme required for effective action in the inhibition of PBP2x. The energetics of hydrogen bonding in the several acyl-enzymes is discussed and comparison is made with carbonyl absorption frequencies of model ethyl esters in a range of organic solvents. A comparison of hydrolytic deacylation with hydroxaminolysis for both chymotryspin and PBP2x leads to the conclusion that deacylation is uncatalysed.