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

Abstract

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.

Substrate specificity of bacterial DD-peptidases (penicillin-binding proteins)

“…There is also evidence from circular dichroism studies in solution that this enzyme changes [130,131]. Infra-red studies of S. pneumoniae PBP2x indicate conformational change on acylation by b-lactams [132]. In view of the above, it was not unreasonable to conclude that the distorted active site structure observed in crystals of PBP2a is responsible for its slow acylation by b-lactams and the b-lactam resistance of bacteria expressing this protein [129], but in view of the situation with S. pneumoniae PBP1b, described above, and the apparent inability of the S. aureus enzyme to catalyze any peptide or depsipeptide hydrolysis in vitro [103], one cannot be completely sure that the distortions observed in the crystal structure are the direct and only cause of the low reactivity of this enzyme in solution.…”

Multiple Conformations of the Acylenzyme Formed in the Hydrolysis of Methicillin by Citrobacter freundii β-Lactamase: a Time-Resolved FTIR Spectroscopic Study

“…Protein perturbation bands are often complex in shape so the relatively simple Gaussian form of the band at 1683 cm -1 argues in favor of assignment to a strongly hydrogen bonded ester carbonyl group conformer. Measurements made on substrates in free solution and model esters suggest that the absorption coefficients of the carbonyl groups in each of the conformers are likely to be similar (4,16,17). Therefore the distribution of the conformers can be calculated from the band areas to give the following: 1737 cm -1 , 10%; 1727 cm -1 , 20%; 1707 cm -1 , 29%; and 1683 cm -1 , 41%.…”

“…This was taken as evidence that catalysis of the hydrolysis of cefoxitin proceeds via an acylenzyme. On the basis of subsequent model studies, performed in our laboratory (4), it may be concluded that this ester carbonyl, whose absorption at 1753 cm -1 decays over a period of several minutes, is poorly, if at all, hydrogen bonded into the oxyanion hole catalytic device. This correlates with cefoxitin having a low deacylation rate and being a very poor substrate for the enzyme with respect to turnover.…”

“…fast but structurally weak methods of UV/vis absorbance and fluorescence spectroscopies (3,4). To our knowledge, there is only a single example in the literature of the application of IR spectroscopy to the study of the -lactamase mechanism.…”

Principles of Enzyme‐Inhibitor Design

“…Unfortunately the simple answer appears to be negative: on-and off-rates and structural rearrangements are difficult to interpret in terms of the energetics of specific interactions. Although detailed spectroscopic studies have begun to shed light on these complex mechanisms (e.g., [14,15]), much more work will be required before all the enthalpic and entropic effects can be unscrambled.…”