title: Spectroscopic and mechanistic studies of heterodimetallic forms of metallo-β-lactamase NDM-1.
abstract: 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.
authors.affiliation: Department of Chemistry and Biochemistry, Miami University , Oxford, Ohio 45056, United States.
authors.forename: Hao
authors.initials: H
authors.lastname: Yang
authors.forename: Mahesh
authors.initials: M
authors.lastname: Aitha
authors.forename: Amy R
authors.initials: AR
authors.lastname: Marts
authors.forename: Alyssa
authors.initials: A
authors.lastname: Hetrick
authors.forename: Brian
authors.initials: B
authors.lastname: Bennett
authors.forename: Michael W
authors.initials: MW
authors.lastname: Crowder
authors.forename: David L
authors.initials: DL
authors.lastname: Tierney
mesh_terms: D004578:Electron Spin Resonance Spectroscopy; D009682:Magnetic Resonance Spectroscopy; D013056:Spectrophotometry, Ultraviolet; D056928:X-Ray Absorption Spectroscopy; D001618:beta-Lactamases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001618:beta-Lactamases; C552864:beta-lactamase NDM-1
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