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Title: Biochemical, Mechanistic, and Spectroscopic Characterization of Metallo-β-lactamase VIM-2

Our 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 also 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.
 [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [2] ;  [3] ;  [1] ;  [1] ;  [1]
  1. Miami Univ., Oxford, OH (United States)
  2. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  3. Louis Stokes Cleveland Dept. of Veterans Affairs Medical Center, Cleveland, OH (United States); Case Western Reserve Univ., Cleveland, OH (United States)
Publication Date:
OSTI Identifier:
Report Number(s):
Journal ID: ISSN 0006-2960
DOE Contract Number:
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry; Journal Volume: 53; Journal Issue: 46
American Chemical Society (ACS)
Research Org:
Brookhaven National Laboratory (BNL), Upton, NY (United States)
Sponsoring Org:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
Country of Publication:
United States