Aitha Mahesh, Marts Amy R, Bergstrom Alex, Møller Abraham Jon, Moritz Lindsay, Turner Lucien, Nix Jay C, Bonomo Robert A, Page Richard C, Tierney David L, Crowder Michael W
Department of Chemistry and Biochemistry, Miami University , 650 East High Street, Oxford, Ohio 45056, United States.
Biochemistry. 2014 Nov 25;53(46):7321-31. doi: 10.1021/bi500916y. Epub 2014 Nov 13.
This study examines metal binding to metallo-β-lactamase VIM-2, demonstrating the first successful preparation of a Co(II)-substituted VIM-2 analogue. Spectroscopic studies of the half- and fully metal loaded enzymes show that both Zn(II) and Co(II) bind cooperatively, where the major species present, regardless of stoichiometry, are apo- and di-Zn (or di-Co) enzymes. We determined the di-Zn VIM-2 structure to a resolution of 1.55 Å, and this structure supports results from spectroscopic studies. Kinetics, both steady-state and pre-steady-state, show that VIM-2 utilizes a mechanism that proceeds through a very short-lived anionic intermediate when chromacef is used as the substrate. Comparison with other B1 enzymes shows that those that bind Zn(II) cooperatively are better poised to protonate the intermediate on its formation, compared to those that bind Zn(II) non-cooperatively, which uniformly build up substantial amounts of the intermediate.
本研究考察了金属与金属β-内酰胺酶VIM-2的结合情况，首次成功制备了钴（II）取代的VIM-2类似物。对半金属负载和全金属负载酶的光谱研究表明，锌（II）和钴（II）均以协同方式结合，无论化学计量比如何，主要存在的物种是脱辅基和二锌（或二钴）酶。我们将二锌VIM-2结构解析到了1.55 Å的分辨率，该结构支持光谱研究的结果。稳态和预稳态动力学均表明，当以头孢西丁为底物时，VIM-2利用一种通过非常短暂的阴离子中间体的机制。与其他B1酶的比较表明，与非协同结合锌（II）的酶相比，协同结合锌（II）的酶在中间体形成时更易于将其质子化，非协同结合锌（II）的酶会均匀积累大量中间体。
