Structural and kinetic studies on metallo-β-lactamase IMP-1
| Autor: | Timothy K. Richmond, Dionne H. Griffin, Michael W. Crowder, David L. Tierney, Abraham Jon Moller, Carlo Sanchez, Robert M. Breece, Brian Bennett |
|---|---|
| Rok vydání: | 2011 |
| Předmět: |
Cations
Divalent Reaction intermediate Biochemistry beta-Lactamases Divalent Catalysis law.invention Metal law Escherichia coli Nitrocefin Electron paramagnetic resonance Serratia marcescens chemistry.chemical_classification Chemistry Hydrolysis Electron Spin Resonance Spectroscopy Cobalt Crystallography Zinc Enzyme X-Ray Absorption Spectroscopy Yield (chemistry) visual_art visual_art.visual_art_medium Spectrophotometry Ultraviolet |
| Zdroj: | Biochemistry. 50(42) |
| ISSN: | 1520-4995 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|