Multiple Conformations of the Acylenzyme Formed in the Hydrolysis of Methicillin by Citrobacter freundii -Lact
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- 作者：Alan-Shaun Wilkinson ; Simon Ward ; Malgosia Kania ; Malcolm G. P. Page ; and Christopher W. Wharton
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：March 30, 1999
- 年：1999
- 卷：38
- 期：13
- 页码：3851 - 3856
- 全文大小：64K
- 年卷期：v.38,no.13(March 30, 1999)
- ISSN：1520-4995
文摘
Time-resolved infrared difference spectroscopy has been used to show that the carbonyl groupof the acylenzyme reaction intermediate in the Citrobacter freundii 
-lactamase-catalyzed hydrolysis ofmethicillin can assume at least four conformations. A single-turnover experiment shows that all fourconformations decline during deacylation with essentially the same rate constant. The conformers arethus in exchange on the reaction time scale, assuming that deacylation takes place only from theconformation which is most strongly hydrogen bonded or from a more minor species not visible in theseexperiments. All conformers have the same (10 cm-1) narrow bandwidth compared with a model ethylester in deuterium oxide (37 cm-1) which shows that all conformers are well ordered relative to freesolution. The polarity of the carbonyl group environment in the conformers varies from 'ether-like' tostrongly hydrogen bonding (20 kJ/mol), presumably in the oxyanion hole of the enzyme. From theabsorption intensities, it is estimated that the conformers are populated approximately proportional to thehydrogen bonding strength at the carbonyl oxygen. A change in the difference spectrum at 1628 cm-1consistent with a perturbation (relaxation) of protein -sheet occurs slightly faster than deacylation.Consideration of chemical model reactions strongly suggests that neither enamine nor imine formation inthe acyl group is a plausible explanation of the change seen at 1628 cm-1. A turnover reaction supportsthe above conclusions and shows that the conformational relaxation occurs as the substrate is exhaustedand the acylenzymes decline. The observation of multiple conformers is discussed in relation to the poorspecificity of methicillin as a substrate of this -lactamase and in terms of X-ray crystallographic structuresof acylenzymes where multiple forms are not apparently observed (or modeled). Infrared spectroscopyhas shown itself to be a useful method for assessment of the uniqueness of enzyme-substrate interactionsin physiological turnover conditions as well as for determination of ordering, hydrogen bonding, andprotein perturbation.
-lactamase-catalyzed hydrolysis ofmethicillin can assume at least four conformations. A single-turnover experiment shows that all fourconformations decline during deacylation with essentially the same rate constant. The conformers arethus in exchange on the reaction time scale, assuming that deacylation takes place only from theconformation which is most strongly hydrogen bonded or from a more minor species not visible in theseexperiments. All conformers have the same (10 cm-1) narrow bandwidth compared with a model ethylester in deuterium oxide (37 cm-1) which shows that all conformers are well ordered relative to freesolution. The polarity of the carbonyl group environment in the conformers varies from 'ether-like' tostrongly hydrogen bonding (20 kJ/mol), presumably in the oxyanion hole of the enzyme. From theabsorption intensities, it is estimated that the conformers are populated approximately proportional to thehydrogen bonding strength at the carbonyl oxygen. A change in the difference spectrum at 1628 cm-1consistent with a perturbation (relaxation) of protein -sheet occurs slightly faster than deacylation.Consideration of chemical model reactions strongly suggests that neither enamine nor imine formation inthe acyl group is a plausible explanation of the change seen at 1628 cm-1. A turnover reaction supportsthe above conclusions and shows that the conformational relaxation occurs as the substrate is exhaustedand the acylenzymes decline. The observation of multiple conformers is discussed in relation to the poorspecificity of methicillin as a substrate of this -lactamase and in terms of X-ray crystallographic structuresof acylenzymes where multiple forms are not apparently observed (or modeled). Infrared spectroscopyhas shown itself to be a useful method for assessment of the uniqueness of enzyme-substrate interactionsin physiological turnover conditions as well as for determination of ordering, hydrogen bonding, andprotein perturbation.