Hydrogen bonding and protein perturbation in beta-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 beta-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-bonded 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.