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.

What vibrations tell about proteins

“…The relationship between stretching frequency and bond strength, DH, has been used to study serine proteases and proteins that are targets for antibiotics (White & Wharton, 1990 ;Chittock et al 1999). These proteins form an acyl-enzyme intermediate, the breakdown of which depends on the interactions with the acyl-enzyme ester carbonyl (for vibrational spectroscopy studies see Tonge & Carey, 1989 ;White & Wharton, 1990).…”

Discrete steps in sensing of β-lactam antibiotics by the BlaR1 protein of the methicillin-resistant Staphylococcus aureus bacterium

“…Spectra of static (nonreacting) mixtures were acquired by using an in situ cuvette with a 100-m pathlength (14). Stopped-flow time-resolved IR data were acquired by using a system derived, with the aim of reducing the dead volume and mixing dead time, from that described previously (18).…”

“…We have used IR spectroscopy previously to evaluate the acylation of the Streptococcus pneumoniae PBP 2x sensitive target transpeptidase by a range of ␤-lactam antibiotics and to evaluate ␤-lactam acylation of the class C ␤-lactamase from Citrobacter freundii. In both examples, one (or more) acyl-protein intermediates were detected, and the conformational changes that accompanied these reactions were observed (14)(15)(16)(17). Herein we report the application of stoppedflow Fourier-transformed IR spectroscopy (18,19) to the simultaneous observation of the rapid reaction kinetics, the evaluation of the structure of the reaction intermediates, and the interpretation of the induced conformational changes accompanying the reaction of the BlaR1 receptor with a ␤-lactam.…”

“…The presence of carbonylstabilizing hydrogen bonds, provided by two amides of the protein backbone (the so-called oxyanion hole), is seen in the crystal structures of both ␤-lactamase and PBP acyl-proteins. A representative value for the carbonyl frequency of these acylproteins is the 1,712-cm Ϫ1 absorption seen for the acyl-protein of S. pneumoniae PBP2x, a ␤-lactam-sensitive transpeptidase that is rapidly acylated by ␤-lactam antibiotics (14). The 1,732-cm Ϫ1 absorbance seen here indicates that the BlaR S acyl-protein is less strongly hydrogen bonded or is in a less polar environment (having a dielectric constant of Ϸ55, as compared with the value of 80 for water).…”

“…Consistent with these structures, the IR spectrum of the BlaR S acyl-protein indicates a single and well ordered conformation, as indicated by its narrow bandwidth (13 cm Ϫ1 ). A representative value for the esters resulting from ␤-lactam solvolysis is an absorption frequency of 1,727 cm Ϫ1 and a bandwidth of 35 cm Ϫ1 in D 2 O solution (14).…”