Display Full Text | Download Full Text
1.47 MB PDF file

Degree

Master of Science, Miami University, Chemistry, 2011.

Abstract

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.

Subject Headings

Biochemistry

Keywords

metallo-β-lactamase; IMP-1

Committee / Advisors

Michael Crowder (Advisor)
Christopher Makaroff (Committee Chair)
David Tierney (Committee Member)
Richard Taylor (Committee Member)

Document number: miami1316402162
Permalink: http://rave.ohiolink.edu/etdc/view?acc_num=miami1316402162

This ETD has been downloaded 58 times (through March 2013)