| Autor: |
Griffin DH; Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056, United States., Richmond TK, Sanchez C, Moller AJ, Breece RM, Tierney DL, Bennett B, Crowder MW |
| Jazyk: |
angličtina |
| Zdroj: |
Biochemistry [Biochemistry] 2011 Oct 25; Vol. 50 (42), pp. 9125-34. Date of Electronic Publication: 2011 Sep 28. |
| DOI: |
10.1021/bi200839h |
| Abstrakt: |
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: |
MEDLINE |
| Externí odkaz: |
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