title: Multiple conformations of the acylenzyme formed in the hydrolysis of methicillin by Citrobacter freundii beta-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.
authors: School of Biochemistry, University of Birmingham, U.K.
authors: A S
authors: AS
authors: Wilkinson
authors: S
authors: S
authors: Ward
authors: M
authors: M
authors: Kania
authors: M G
authors: MG
authors: Page
authors: C W
authors: CW
authors: Wharton
mesh_terms: D001426:Bacterial Proteins; D002264:Carboxylic Acids / Q000737:chemistry / Q000378:metabolism; D016971:Citrobacter freundii / Q000201:enzymology*; D006860:Hydrogen Bonding; D006868:Hydrolysis; D007700:Kinetics; D008712:Methicillin / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D017550:Spectroscopy, Fourier Transform Infrared; D001618:beta-Lactamases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D002264:Carboxylic Acids; C048795:AmpC beta-lactamases; D001618:beta-Lactamases; D008712:Methicillin