This work is aimed at developing an understanding of enzyme mechanisms through the design of transition state analogs, that resemble activated intermediates in substrate transformations catalyzed by enzymes. Because transition state analogs are chemically stable and cannot collapse to form products, their affinities for enzymes persist, and they are exceptionally potent metabolic inhibitors. In addition, structural observations on their complexes with enzymes can reveal the origins of their unusual affinities for enzymes, which parallel the interactions that are responsible for the catalytic rate enhancements that enzymes bring about. Targets proposed for inhibitor design include three enzymes involved in purine biosynthesis: adenylosuccinate synthetase, inosinic acid dehydrogenase and guanylate synthetase (XMP aminase); in each of the cases there is evidence of a high-energy intermediate whose structure can in principle be imitated with one or more stable analogs that we intend to prepare. Bisubstrate analogs are to be prepared for three kinases that are active in rapidly-dividing cells: thymidine kinase, thymidylate kinase and tyrosine-specific protein kinases. In view of evidence that pyridoxal-requiring enzymes proceed through planar intermediates, a planar bisubstrate analog is to be prepared as a potential inhibitor of pyridoxamine pyruvate transaminase. Finally, experiments relating to the production and purification of new enzyme activities are proposed, using transition state analogs as templates and as eluting agents for chromatographic separation of molecules according to their catalytic turnover numbers.
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