Amino acids are central to the many metabolic processes that sustain living cells and organisms. The long-term objectives of this research endeavor are to uncover and elucidate new metabolic reactions on the enzymic level involving 4-hydroxyglutamic acid/2- keto-4-hydroxyglutaric acid and threonine, and also to examine the structure/function interrelationships of key enzymes involved in order to establish structural requirements for catalytic and/or regulatory activity. Specifically, in vitro enzyme studies will be carried out to ascertain how 4-hydroxyglutamic acid is converted to glutamic acid in mammals and also to determine how 2-keto-4-hydroxyglutaric acid is utilized by Acetobacter suboxydans, an organism that lacks the conventional Krebs cycle, to biosynthesize di- and tricarboxylic acids. New metabolic intermediates and enzymes involved will be isolated and characterized. Three enzymes, namely, 2-keto-4- hydroxyglutarate aldolase, L-threonine dehydrogenase, and 2-amino- 3-ketobutyrate which we have succeeded in isolating in pure form and substantial quantities, will be the objects of in-depth structure/function studies. Selective chemical modification of these enzymes will be carried out to determine specific amino acid residues that are required for catalytic activity, peptides containing these residues will be separately isolated and sequenced by HPLC methodologies, and their position in the complete primary structure of the enzyme established. The genes for these enzymes will be cloned, selected amino acid. changes in the structure of the enzymes made by site-directed mutagenesis procedures, and the effects of such changes on their catalytic/regulatory properties determined. The aldolase catalyzes a key reaction in the mammalian metabolism of hydroxyproline, the dehydrogenase in the biosynthesis of Vitamin B12 by microorganisms and the formation of aminoacetone (which is excreted by humans), and the dehydrogenase/ lyase combination initiates a new route for serine biosynthesis. The goal is to understand metabolic and enzymic processes since life is dependent on an organized and balanced system of enzymes and since more and more diseased states have fundamental molecular and/or enzymic facets.
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