The principal goal of this project is to elucidate the molecular factors that lead to drug resistance in the B-lactamase family of enzymes. (-lactamases are produced by bacteria and they destroy penicillin-type molecules before they can kill their bacterial targets. Thus, B-lactamases themselves are targets for inhibitors. Clinical isolates demonstrate that, B-lactamases develop resistance to their drug-inhibitors by undergoing point mutations. This proposal will elucidate the chemical reactions between a SHV-1 B-lactamase and the clinically important drugs, tazobactam, sulbactam and clavulanic acid; these compounds act as """"""""suicide inhibitors."""""""" The reactions will be characterized by Raman crystallography - by following the reactions in single crystals of the enzyme using a Raman microscope. The drugs are injected separately into the mother liquor containing a crystal of B-lactamase, the inhibitors diffuse fully into the crystal in less than one minute and the subsequent reaction in the active site can be followed via the Raman difference spectrum. Using suitable forms of the enzyme, the structures and populations of the Michaelis complexes, acyl enzymes and final products can be defined in the crystals from the Raman data. By comparing these properties for the wild-type enzyme, and the enzyme that has developed a resistance to the inhibitors, unique insight into the molecular mechanisms underlying resistance will be gained. The resistant forms of the B-lactamase are M691, M69L and M69V, selected for their clinical relevance. The ability of the Raman method to characterize populations of intermediates in single crystals will be used to select optimal times for flash freezing. The crystals containing the trapped reaction intermediates will then be characterized by X-ray crystallography. For the class D B-lactamases OXA-10 and OXA-1, recent studies have indicated that a carbamylated lysine plays a key role in active site chemistry. Raman crystallography will be used to confirm this novel and controversial finding.
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