Multiple conformations of the acylenzyme formed in the hydrolysis of methicillin by Citrobacter freundii beta-lactamase: a time-resolved FTIR spectroscopic study

Time-resolved infrared difference spectroscopy has been used to show that t he carbonyl group of the acylenzyme reaction intermediate in the Citrobacte r 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 co nstant. The conformers are thus in exchange on the reaction time scale, ass uming 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 com pared with a model ethyl ester in deuterium oxide (37 cm(-1)) which shows t hat all conformers are well ordered relative to free solution. The polarity of the carbonyl group environment in the conformers varies from 'ether-lik e' to strongly hydrogen bonding (20 kJ/mol), presumably in the oxyanion hol e 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 16 28 cm(-1) consistent with a perturbation (relaxation) of protein beta-sheet occurs slightly faster than deacylation. Consideration of chemical model r eactions 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 conform ational relaxation occurs as the substrate is exhausted and the acylenzymes decline, The observation of multiple conformers is discussed in relation t o the poor specificity of methicillin as a substrate of this beta-lactamase and in terms of X-ray crystallographic structures of acylenzymes where mul tiple 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 perturbati on.