MECHANISTIC STRUCTURE-FUNCTION-INHIBITOR STUDIES

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Project I: The incidence of diabetes and obesity is increasing at an alarming rate, affecting millions around the world. Human pancreatic alpha-amylase (HPA; 496 a.a.'s) is a unique and compelling target for the development of potential therapeutics for both these diseases, given its central role in starch digestion. However, successful development of such therapeutics is dependent on a structural understanding of how the catalytic residues of HPA function and the mode of substrate binding in the elongated binding cleft present. Although we have successfully applied site directed mutagenesis techniques and grown crystals of both wild-type and variant proteins, interpretation of the structural results of complexes formed by substrates and inhibitors has had limited success due to a lack of resolution (~2.0 ¿) using our home laboratory x-ray source (Rigaku RU-300). Access to the SSRL data collection facility will greatly enhance the resolutions of our structural studies, thereby allowing for a comprehensive interpretation of such mechanistic complexes and facilitating the development of novel therapeutics based on this mechanistic data. Project II: Surprisingly, only Gram-negative bacteria contain hexameric Type II citrate synthases that have the special property of being metabolically regulated. In contrast, the dimeric citrate synthases of other organisms (including humans) is unregulated. Since many Gram-negative bacteria are dangerous human pathogens, the special properties of Type II citrate synthases could form a basis for the development of novel anti-microbials. Key to the development of such anti-microbials is the structural characterization of a hexameric Type II citrate synthase (CS) in its various allosteric and metabolically controlled states.