During the period of support requested in this application, we plan to continue our efforts to understand the chemical mechanism of action of several alkyl transfer enzymes. We plan to extend our research into two additional group transfer reactions involving glutamine and asparagine derivatives. Specifically, we will continue to study appropriate non-enzymic """"""""model reactions"""""""" in order to understand more fully the details of general catalysis of these reactions. We will investigate the effect of changing the nucleophile and leaving group on the position of the transition state on the reaction coordinate, and the effect such changes have on the degree of proton transfer in the transition state. These model reactions are designed to mimic enzyme-catalyzed O-alkylation, N-alkylation, and S-alkylation by compounds such as S-adenosylmethionine (O,N,S), and methyltetrahydrofolate (S). We will continue to probe the mechanism of enzyme-catalyzed alkyl transfer reactions by studying the stereochemistry of the bacterial and mammalian aminopropyltransferases, spermidine synthase and spermine synthase, and a bacterial methionine synthase (B12-independent). We will study the transport of [35S]-nucleoside 5'-thioethers in cultured mammalian cells in order to address quantitatively the extent to which metabolically stable analogs of 5'-substituted nucleosides are transported across cell membranes. We will initiate studies on the mechanism of action of two enzymes involved in important, but poorly understood, glutamate and aspartate biochemistry. Specifically, we will study the mechanism of action of folylpolyglutamate synthetase, the enzyme involved in the biosynthesis of so-called folate conjugates, the predominant intracellular form of folates. In addition, we will initiate mechanistic studies on the unusual reaction by which asparagine-linked oligosaccharides are biosynthesized. These mechanistic studies on polyglutamate and glycoprotein biosynthetic enzymes will employ non-kinetic probes of the reactions.
| Banerjee, R V; Shane, B; McGuire, J J et al. (1988) Dihydrofolate synthetase and folylpolyglutamate synthetase: direct evidence for intervention of acyl phosphate intermediates. Biochemistry 27:9062-70 |
| Coward, J K (1988) Aminopropyltransferases: mechanistic studies and the synthesis of specific inhibitors. Adv Exp Med Biol 250:111-5 |
