Spectroscopic and mechanistic studies of heterodimetallic forms of metallo-β-lactamase NDM-1. J Am Chem Soc 2014 May 21;136(20):7273-85
Date
04/24/2014Pubmed ID
24754678Pubmed Central ID
PMC4046764DOI
10.1021/ja410376sScopus ID
2-s2.0-84901049657 (requires institutional sign-in at Scopus site) 60 CitationsAbstract
In an effort to characterize the roles of each metal ion in metallo-β-lactamase NDM-1, heterodimetallic analogues (CoCo-, ZnCo-, and CoCd-) of the enzyme were generated and characterized. UV-vis, (1)H NMR, EPR, and EXAFS spectroscopies were used to confirm the fidelity of the metal substitutions, including the presence of a homogeneous, heterodimetallic cluster, with a single-atom bridge. This marks the first preparation of a metallo-β-lactamase selectively substituted with a paramagnetic metal ion, Co(II), either in the Zn1 (CoCd-NDM-1) or in the Zn2 site (ZnCo-NDM-1), as well as both (CoCo-NDM-1). We then used these metal-substituted forms of the enzyme to probe the reaction mechanism, using steady-state and stopped-flow kinetics, stopped-flow fluorescence, and rapid-freeze-quench EPR. Both metal sites show significant effects on the kinetic constants, and both paramagnetic variants (CoCd- and ZnCo-NDM-1) showed significant structural changes on reaction with substrate. These changes are discussed in terms of a minimal kinetic mechanism that incorporates all of the data.
Author List
Yang H, Aitha M, Marts AR, Hetrick A, Bennett B, Crowder MW, Tierney DLAuthor
Brian Bennett D.Phil. Professor and Chair in the Physics department at Marquette UniversityMESH terms used to index this publication - Major topics in bold
Electron Spin Resonance SpectroscopyMagnetic Resonance Spectroscopy
Spectrophotometry, Ultraviolet
X-Ray Absorption Spectroscopy
beta-Lactamases
