KINETIC AND SPECTROSCOPIC STUDIES OF L1, THE METALLO-?-LACTAMASE FROM Stenotrophomonas maltophilia
Metallo-?-lactamase L1 (m?l L1), originally from Stenotrophomonas maltophilia, can hydrolyze all ?-lactam containing antibiotics. Previous crystal structures showed that m?l L1binds two Zn(II) ions in the active site, and there is a long flexible loop above the metal center. To better understand the function of the Zn(II) ions in m?l L1, several metal-substituted and heterobimetallic analogs of L1 were generated and characterized using spectroscopic and kinetic studies. The metal binding sites in L1 can accommodate a number of different metal ions to afford catalytically-active analogs. Pre-steady state kinetic studies using nitrocefin as substrate showed that both Zn(II) ions were required for the formation of intermediate and that an analog of L1 containing only one equivalent of Zn(II) is slightly active. Different metal ions in the Zn2 metal binding site modulated m?l L1's substrate preference, and L1 analogs containing Ni(II) or Fe in the Zn2 site were unable to hydrolyze penicillins but could hydrolyze cephalosporins and carbapenems. Rapid freeze quench (RFQ) EPR and 1H NMR spectra confirmed the catalytic function of both metal sites. Based on the kinetic and spectroscopic studies, a reaction mechanism of m?l L1 was proposed when nitrocefin is the substrate. In an effort to understand the function of the loop above the active site, fluorescence resonance energy transfer (FRET) and double electron electron resonance (DEER) studies were proposed. The preparation of double FRET labeled L1 analogs was unsuccessful; however, a double spin-labeled analog was made. Preliminary RFQ-DEER data show that this technique can be used to probe intramolecular motions during catalysis. The data from this dissertation can be used to guide future rational inhibitor design efforts.
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:metallo ? lactamase zn ii spectroscopic kinetic enzyme mechanism
Date of Publication:01/01/2008