The proposed research involves a combination of method development for the acquisition of structural information about enzyme-substrate or enzyme-inhibitor complexes, and site-directed mutagenesis studies on B-lactamase to determine the role of active-site residues in catalysis and inhibition. Fourier Transfer Infrared (FTIR) spectroscopy, especially attenuated total reflectance (ATR) FTIR, will be used to study enzyme-substrate intermediates (stabilized at low temperatures) and enzyme-inhibitor (e.g. transition state analogs) complexes. FTIR has the potential to reveal not only new bonds formed in enzyme-ligand complexes, but also bond deformations and polarization, as well as conformational changes in the enzyme. These methods will be applied to several systems to increase our understanding of the molecular basis for enzyme catalysis and inhibition . Resistance of pathogenic organisms toward B-lactam antibiotics is a major international health problem. In the majority of cases the reason for the resistance is the presence of B-lactamases produced by the infectious agent, which hydrolyze the B-lactam rendering it ineffective as a antibiotic . This research is aimed at increasing our understanding of the mechanism of catalysis of B-lactamases.m Site-directed mutagenesis will be used to determine the role of several residues which have been proposed to be important in catalysis. The ultimate goal is the design of mechanism- based inhibitors of the B-lactamases which will also function as antibiotics.
