A series of spectoscopic, chemical and crystallographic studies will be conducted on three highly purified vitamin B6-dependent enzymes: cytosolic aspartate aminotransferase of pig hearts (AspAT), tryptophanase of Escherichia coli and glutamate decarboxylase of E. coli. The goals will be to provide understanding of basic mechanisms of pyridoxal phosphate- dependent catalysis and to assist in the design of useful enzyme-activated inhibitors. (1) We will conduct a series of microspectrophotometric measurements on crystals of aspartate aminotransferase. We will measure binding constants within crystals for inhibitors and natural substrates. (2) We will collaborate in determining structures of substrate and inhibitor complexes of this enzyme. We will make special efforts to stabilize the erythro-3-hydroxyaspartate complex and to deduce its structure. (3) We will separate individual species of the self-inactivated beta subform of AspAT by isoelectric focusing and will crystallize at least the major species and some other less abundant forms. This homogeneous beta form will be analyzed by X-ray diffraction and will be used by us in microspectrophotometry for a variety of experiments. We will characterize an addition major compound released from the inactivated subunit of the beta subform by heating. (4) The 6-fluoro derivatives of pyridoxal and pyridoxamine phosphates, incorporated in to the active sites of AspAT, tryptophanase, and bacterial glutamate decarboxylase, will be studied by 19F NMR spectroscopy. (5) We will try to crystallize AspAT inhibited by enzyme-activated inhibitors including beta-methyleneaspartate, serine sulfate, and acetylenic GABA. This should allow us to see directly the structures of different types of inhibited enzyme. The resulting knowledge will be important for rational design of drugs for inhibition of other transaminases. We also propose to do modeling of inhibitor action using an Evans & Sutherland graphics terminal. We will try series of spectroscopic studies on tryptophanase and glutamate decarboxylase aimed at elucidating the structures of intermediate species. We will also attempt to obtain crystals of these enzymes suitable for X-ray studies. (7) We will conduct nonenzymic studies on formation of Al3+ -stabilized quinonoid species in methanol and we will compare rates of alpha hydrogen removal in model systems with pyridoxal phosphate, its N-methyl derivatives and with 6-fluoropyridoxal phosphate.
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