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

Abstract

Time-resolved infrared difference spectroscopy has been used to show that the carbonyl group of the acylenzyme reaction intermediate in the Citrobacter freundii beta-lactamase-catalyzed hydrolysis of methicillin can assume at least four conformations. A single-turnover experiment shows that all four conformations decline during deacylation with essentially the same rate constant. The conformers are thus in exchange on the reaction time scale, assuming that deacylation takes place only from the conformation which is most strongly hydrogen bonded or from a more minor species not visible in these experiments. All conformers have the same (10 cm-1) narrow bandwidth compared with a model ethyl ester in deuterium oxide (37 cm-1) which shows that all conformers are well ordered relative to free solution. The polarity of the carbonyl group environment in the conformers varies from 'ether-like' to strongly hydrogen bonding (20 kJ/mol), presumably in the oxyanion hole of the enzyme. From the absorption intensities, it is estimated that the conformers are populated approximately proportional to the hydrogen bonding strength at the carbonyl oxygen. A change in the difference spectrum at 1628 cm-1 consistent with a perturbation (relaxation) of protein beta-sheet occurs slightly faster than deacylation. Consideration of chemical model reactions strongly suggests that neither enamine nor imine formation in the acyl group is a plausible explanation of the change seen at 1628 cm-1. A turnover reaction supports the above conclusions and shows that the conformational relaxation occurs as the substrate is exhausted and the acylenzymes decline. The observation of multiple conformers is discussed in relation to the poor specificity of methicillin as a substrate of this beta-lactamase and in terms of X-ray crystallographic structures of acylenzymes where multiple forms are not apparently observed (or modeled). Infrared spectroscopy has shown itself to be a useful method for assessment of the uniqueness of enzyme-substrate interactions in physiological turnover conditions as well as for determination of ordering, hydrogen bonding, and protein perturbation.

Three Decades of β-Lactamase Inhibitors

“…The inhibition of NMC-A by the monobactam derivative 13 ( Fig. 1) is enhanced by the incorporation of chemical components that confer structural flexibility to the inhibitor, allowing the enzyme-substrate species to adopt multiple conformations and trap the enzyme in energy minima unfavorable for hydrolysis (273,308,445). Another viable approach is to design inhibitors that induce conformational changes in the enzyme or displace key water molecules important for hydrolysis, such as seen with the large seven-membered intermediate formed from the methylidene penems (60,294,429).…”

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

“…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.…”

Inhibition of the Broad Spectrum Nonmetallocarbapenamase of Class A (NMC-A) β-Lactamase from Enterobacter cloacae by Monocyclic β-Lactams

“…Molecular dynamics simulations have underscored the existence of considerable structural flexibility for the inhibited enzyme species, a process that allowed for the observations of the two species seen in the x-ray structure of the inhibited enzyme as important entities in the course of simulations. This structural dynamic nature is probably present in other acyl-enzyme intermediates for ␤-lactamases such as recently documented for another ␤-lactamase (25). Since simulations indicate the possibility of such motion in picosecond time scale and the fact that catalysis by these enzymes take place on millisecond time scale, it is likely that such dynamic motion takes place with typical substrates for these enzymes as well.…”