CAUSE AND EFFECT OF DIMER ASYMMETRY IN MERCURIC REDUCTAS

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The proposed research lies in the area of mechanistic enzymology, with long term goals of elucidating how protein structural elements or motifs specifically contribute to catalytic efficiency. Of particular interest is the study of molecular mechanisms and functional consequences of communication between remote ligand binding sites in proteins. For a wide variety of receptors, enzymes and binding proteins, signalling between distant sites is critical to the physiological function. Consequently, disruptions of site-site communication result in defective protein function, which almost certainly cause disease states if the defect is severe. Thus, identifying structural motifs and/or molecular networks for different types of intersite communication could provide valuable insight into disease-causing mutations, as well as suggest alternative strategies for designing drugs to control protein function. From a mechanistic view, the most poorly understood type of intersite communication is negative cooperativity between identical sites on homooligomers. In an alternating sites hypothesis, originally proposed for ATP synthases and, more recently for mercuric reductase, the functional role ascribed to negative cooperativity is one of switching the affinity for ligands at identical sites of a homooligomer in a concerted fashion to facilitate different steps of a catalytic pathway. Substantial evidence supports this hypothesis in the ATP synthases, but structural studies are difficult in that system. By contrast, mercuric reductases are soluble homodimers; genes for enzyme from two sources are overexpressed; and the crystal structure of one of those has been solved, all of which make this enzyme an attractive model system to explore the mechanistic significance and molecular basis of homooligomer asymmetry resulting from negative cooperative communication. Specifically, the predictions of the alternating sites hypothesis, as described for mercuric reductase will be tested using a combination of equilibrium titrations and single turnover kinetics on a set of mutants lacking one or more ligands in the Hg(II) binding sites. Additionally, spectrally characterized Hg(II) complexes of wild type and mutant enzymes will be structurally characterized using XAFS methods. Crystal structure data will be used to identify possible pathways for communication, which will then be explored through site- directed mutagenesis and complete physical and mechanistic characterization of the novel mutant enzymes.