title: Structural and kinetic studies on metallo-β-lactamase IMP-1.
abstract: In an effort to probe for metal binding to metallo-β-lactamase (MβL) IMP-1, the enzyme was overexpressed, purified, and characterized. The resulting enzyme was shown to bind 2 equiv of Zn(II), exhibit significant catalytic activity, and yield EXAFS results similar to crystallographic data previously reported. Rapid kinetic studies showed that IMP-1 does not stabilize a nitrocefin-derived reaction intermediate; rather, the enzyme follows a simple Michaelis mechanism to hydrolyze nitrocefin. Metal-substituted and metal-reconstituted analogues of IMP-1 were prepared by directly adding metal ion stocks to metal-free enzyme, which was generated by dialysis versus EDTA. UV-vis studies on IMP-1 containing 1 equiv of Co(II) showed a strong ligand-to-metal charge transition at  340 nm, and the intensity of this feature increased when the second equivalent of Co(II) was added to the enzyme. EXAFS fits on IMP-1 containing 1 equiv of Co(II) strongly suggest the presence of a metal-metal interaction, and EPR spectra of the IMP-1 containing 1 and 2 equiv of Co(II) are very similar. Taken together, steady-state kinetic and spectroscopic studies suggest that metal binding to metal-free IMP-1 follows a positive-cooperative mode.
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056, United States.
authors.forename: Dionne H
authors.initials: DH
authors.lastname: Griffin
authors.forename: Timothy K
authors.initials: TK
authors.lastname: Richmond
authors.forename: Carlo
authors.initials: C
authors.lastname: Sanchez
authors.forename: Abraham Jon
authors.initials: AJ
authors.lastname: Moller
authors.forename: Robert M
authors.initials: RM
authors.lastname: Breece
authors.forename: David L
authors.initials: DL
authors.lastname: Tierney
authors.forename: Brian
authors.initials: B
authors.lastname: Bennett
authors.forename: Michael W
authors.initials: MW
authors.lastname: Crowder
mesh_terms: D002413:Cations, Divalent; D003035:Cobalt / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006868:Hydrolysis; D012706:Serratia marcescens / Q000201:enzymology* / Q000235:genetics; D013056:Spectrophotometry, Ultraviolet; D056928:X-Ray Absorption Spectroscopy; D015032:Zinc / Q000737:chemistry; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D002413:Cations, Divalent; D003035:Cobalt; C409201:beta-lactamase IMP-1; D001618:beta-Lactamases; D015032:Zinc