We propose to continue collaboration with Dr. Arthur Arnone at the University of Iowa and with Dr. Marvin Makinen at the University of Chicago on X-ray diffraction and polarized optical spectroscopic studies of crystalline cytosolic aspartate aminotransferase (glutamate-oxaloacetate transaminase) of pig hearts.
The aim will be to understand the dynamic behavior of the coenzyme and protein during various stages of catalysis by this important heart enzyme. Among the features to be studied is a major conformational change of the protein that accompanies the binding of substrates or inhibitors. We will attempt to precisely define the details of the catalytic process and the role of the protein as a """"""""solvent"""""""" for the reaction. We will continue to study the action of serine sulfate, Beta-methyleneaspartate, vinylglycine and other """"""""suicide inactivators"""""""" of vitamin B6-dependent enzymes. We will prepare crystalline derivatives with which it should be possible to see directly the structure of three different types of inhibited enzyme. This knowledge will be important for rational design of drugs for inhibition of other transaminases. We will attempt to determine the structure of the inhibited active site of the naturally occurring Beta subform which is only partially active. This will involve characterization of compounds released by heating the inactivated subunits and isolation and chemical characterization of peptides carrying a covalently bonded inhibitor or modified substrate. The possible biological and medical significance of the presence in tissues of large amounts of partially inactivated enzyme will be considered. We will continue studies of a series of analogs of pyridoxal phosphate with aspartate aminotransferase and glutamate decarboxylase. Among these are analogs that do not permit transimination and 6-fluropyridoxal phosphate, a compound with interesting electronic properties and with a good NMR signal that can be seen when bound to enzymes. We will study further the formation of fluorescent pyrrolopyridines with pyridoxal 5 foot-sulfate. We will continue nonenzymatic studies of Schiff bases, especially of diamines using both proton NMR and computer-assisted spectroscopic analysis. All of these studies have a direct relevance to a large group of important vitamin B6-dependent enzymes.
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