Document Type

Article

Language

eng

Format of Original

13 p.

Publication Date

5-2014

Publisher

American Chemical Society

Source Publication

Journal of the American Chemical Society

Source ISSN

0002-7863

Original Item ID

doi: 10.1021/ja410376s

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, 1H 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.

Comments

Published version. Journal of the American Chemical Society, Vol. 136, No. 20 (May 2014): 7273–7285. DOI. © 2014 American Chemical Society. Used with permission.

This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.

Brian Bennett was affiliated with Medical College of Wisconsin at the time of publication.

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