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STRUCTURAL and KINETIC STUDIES on METALLO-β-LACTAMASE IMP-1

Griffin, Dionne Hope
http://rave.ohiolink.edu/etdc/view?acc_num=miami1316402162

Abstract Details

2011, Master of Science, Miami University, Chemistry and Biochemistry.
In an effort to probe for metal binding to metallo-β-lactamase (MβL) IMP-1, the enzyme was over-expressed, purified, and characterized. The resulting enzyme was shown to bind 2 equivalents 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 analogs 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 equivalent 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 equivalent of Co(II) strongly suggest the presence of a metal-metal interaction, and EPR spectra of the IMP-1 containing 1 and 2 equivalents of Co(II) are very similar. Taken together, steady-state kinetic and spectroscopic studies strongly suggest that metal binding to metal-free IMP-1 follows a positive-cooperative mode.
Michael Crowder (Advisor)
Christopher Makaroff (Committee Chair)
David Tierney (Committee Member)
Richard Taylor (Committee Member)
Biochemistry
metallo-&946; -lactamase , IMP-1

Recommended Citations

Citations

  • Griffin, D. H. (2011). STRUCTURAL and KINETIC STUDIES on METALLO-β-LACTAMASE IMP-1 [Master's thesis, Miami University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=miami1316402162

    APA Style (7th edition)

  • Griffin, Dionne. STRUCTURAL and KINETIC STUDIES on METALLO-β-LACTAMASE IMP-1. 2011. Miami University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=miami1316402162.

    MLA Style (8th edition)

  • Griffin, Dionne. "STRUCTURAL and KINETIC STUDIES on METALLO-β-LACTAMASE IMP-1." Master's thesis, Miami University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=miami1316402162

    Chicago Manual of Style (17th edition)

miami1316402162
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© 2011, all rights reserved.
This open access ETD is published by Miami University and OhioLINK.