The long term objectives of the research supported by this grant are to determine the chemical mechanisms by which the coenzyme forms of vitamin B1 (thiamin pyrophosphate, TPP) and vitamin B6 (pyridoxal-5'-phosphate, PLP) act in reactions that cannot be explained by the conventional mechanisms known for these coenzyme. The role of 2-acetyl-TPP in the metabolism of pyruvate catalyzed by the pyruvate dehydrogenase (PDH) complex will be determined using chemical methods. Several inherited diseases of pyruvate metabolism are caused by defects in the PDH complex. The standard mechanistic paradigm for the coenzymatic action of PLP involves the stabilization of carbanionic intermediates by the coenzyme. However, in a major focus of research supported by this grant, the action of PLP in the lysine 2,3-aminomutase-catalyzed conversion of lysine of beta-lysine PLP is proposed to act by stabilizing a radical intermediate, rather than a carbanion,. This is a new chemical mechanism for PLP with other aminomutases, such as beta-lysine mutase and arginine 2,3,-aminomutase. The aminomutases catalyze 1,2-amino group migrations that are important in amino acid metabolism and the biosynthesis of antibiotics such as mycomycin and Blasticidin I. Research supported by this grant will test the new mechanistic hypothesis by application of spectroscopic and chemical methods. The aminomutase reactions are also related to vitamin B12; in fact beta-lysine aminomutase is an adenosylcobalamin-dependent enzyme. Lysine 2,3-aminomutase catalyzes a chemically similar 1,2-amino group rearrangement, but does so without a vitamin B12 coenzyme, despite the fat that nearly all rearrangements of this chemical type in living cells involve adenosylcobalamin as an essential coenzyme. The cofactors of lysine 2,3,-aminomutase are iron-sulfur cluster, cobalt(II) and S- adenosylmethionine (AdoMet). Currently available information indicates that AdoMet and the metal cofactors interact chemically to generate an adenosyl- cofactor that has chemical properties in common with adenosycobalamin.a The research supported by this grant will seek to characterize this cofactor by the application of spectroscopic and chemical methods. Thus the central thrust of most of research is the elucidation of new chemical mechanisms in the biological actions of the coenzymatic forms of vitamins B1,B6, and B12. A biochemical problem of recent origin is that of determining the chemical functions of proteins discovered and characterized by genetic methods. A new method for identifying endogenous ligands for such proteins is needed. Such method is being developed for this purposed.
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