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Structure/function studies on metallo-B-lactamase ImiS from Aeromonas bv. sobria

by Sharma, Narayan Prasad.

Abstract (Summary)
STRUCTURE/FUNCTION STUDIES ON METALLO-?-LACTAMASE ImiS FROM Aeromonas bv. sobria by Narayan Prasad Sharma Zinc-containing metallo-?-lactamases(M?Ls) are an emerging class of enzymes that render bacteria resistant to ?-lactam-containing antibiotics, and these enzymes have been grouped into 3 distinct classes, B1, B2 and B3. While extensive structure/mechanistic information is known about the B1 and B3 M?Ls, relatively little is known about the B2 enzymes. In an effort to characterize a B2 M?L, spectroscopic and mechanistic studies on metallo-?-lactamase ImiS from Aeromonas bv. sobria, were performed. 1H NMR, UV-Vis, EPR, and EXAFS spectroscopic studies on Co(II)substituted ImiS revealed that the active site metal ion is tetrahedrally-coordinated by 1 cysteine, 1 histidine, and 1 aspartic acid and presumably 1 water ligands. Steady-state, presteady-state kinetic studies were conducted on ImiS and its reaction with imipenem and meropenem. pH Dependence studies revealed no inflection points in the pH range 5.0-8.5, while proton inventories demonstrated at least 1 rate-limiting proton transfer. Stopped-flow fluorescence, stopped-flow UV-Vis, and rapid freeze quench EPR studies on Co(II)-substituted ImiS revealed a kinetic mechanism in which the rate-limiting step is C-N bond cleavage. Site-directed spin labeling and EPR spectroscopy have revealed the movement of ?-helix in kinetically competent time scale. Taken together, this dissertation offers, the only structural and mechanistic information on a B2 M?L and a common rate-limiting step for all M?L’s that can be used to guide future inhibitor design efforts.
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Advisor:

School:Miami University

School Location:USA - Ohio

Source Type:Master's Thesis

Keywords:metallo b lactamases enzyme kinetics mechanism beta organometallic chemistry aeromonas enzymes

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