Spectroscopic and Mechanistic Studies of Heterodimetallic Forms of Metallo-尾-lactamase NDM-1

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• 作者：Hao Yang ; Mahesh Aitha ; Amy R. Marts ; Alyssa Hetrick ; Brian Bennett ; Michael W. Crowder ; David L. Tierney
• 刊名：Journal of the American Chemical Society
• 出版年：2014
• 出版时间：May 21, 2014
• 年：2014
• 卷：136
• 期：20
• 页码：7273-7285
• 全文大小：525K
• 年卷期：v.136,no.20(May 21, 2014)
• ISSN：1520-5126

文摘

In an effort to characterize the roles of each metal ion in metallo-尾-lactamase NDM-1, heterodimetallic analogues (CoCo-, ZnCo-, and CoCd-) of the enzyme were generated and characterized. UV鈥搗is, 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-尾-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.