Spencer J, Clarke A R, Walsh T R
Department of Pathology and Microbiology, University of Bristol School of Medical Sciences, University Walk, Bristol BS8 1TD, United Kingdom.
J Biol Chem. 2001 Sep 7;276(36):33638-44. doi: 10.1074/jbc.M105550200. Epub 2001 Jul 6.
Stopped-flow tryptophan fluorescence under single turnover and pseudo-first-order conditions has been used to investigate the kinetic mechanism of beta-lactam hydrolysis by the Stenotrophomonas maltophilia L1 metallo-beta-lactamase. For the cephalosporin substrates nitrocefin and cefaclor and the carbapenem meropenem, a substantial quench of fluorescence is observed on association of substrate with enzyme. We have assigned this to a rearrangement event subsequent to formation of an initial collision complex. For the colorimetric compound nitrocefin, decay of this dark inter- mediate represents the overall rate-determining step for the reaction and is equivalent to decay of a previously observed state in which the beta-lactam amide bond has already been cleaved. For both cefaclor and meropenem, the rate-determining step for hydrolysis is loss of a second, less quenched state, in which, however, the beta-lactam amide bond remains intact. We suggest, therefore, that the mechanism of hydrolysis of nitrocefin by binuclear metallo-beta-lactamases may be atypical and that cleavage of the beta-lactam amide bond is the rate-determining step for breakdown of the majority of beta-lactam substrates by the L1 enzyme.
在单周转和准一级条件下的停流色氨酸荧光已被用于研究嗜麦芽窄食单胞菌L1金属β-内酰胺酶催化β-内酰胺水解的动力学机制。对于头孢菌素底物硝基头孢菌素和头孢克洛以及碳青霉烯类美罗培南，在底物与酶结合时观察到荧光的显著淬灭。我们将此归因于初始碰撞复合物形成后的重排事件。对于比色化合物硝基头孢菌素，这种暗中间体的衰减代表了反应的总体速率决定步骤，并且等同于先前观察到的β-内酰胺酰胺键已经断裂的状态的衰减。对于头孢克洛和美罗培南，水解的速率决定步骤是第二种淬灭程度较小的状态的消失，然而，其中β-内酰胺酰胺键仍然完整。因此，我们认为双核金属β-内酰胺酶催化硝基头孢菌素水解的机制可能是非典型的，并且β-内酰胺酰胺键的断裂是L1酶催化大多数β-内酰胺底物分解的速率决定步骤。
