Uridine diphospho-glucose and -galactose are key intermediates in the biosynthesis of complicated poly-saccharides and in the transfer of glycosyl groups to a variety of other acceptors. Our primary objective with this proposal is to elucidate the mechanism of action for two enzymes involved in the synthesis of UDP-sugars and two enzymes that utilize these compounds in the transfer of sugar residues to various acceptor molecules. Toward this end we will be further developing and enhancing the technique of positional isotope exchange for determining enzyme reaction mechanisms. This technique can be used with any enzymatic reaction in which nonequivalent functional groups of the substrate become rotationally or torsionally equivalent during the transformation to products. We will show that this technique is capable of determining the partitioning of enzyme complexes, establishing kinetic mechanisms, and measuring microscopic rate constants for the dissociation of ligands from central enzyme complexes. The biosynthesis of UDP-sugars by the enzymes galactose-1-phosphate uridylyltransferase and UDP-glucose pyrophosphorylase will be analyzed via this technique. The mechanisms for the transfer of glycosyl groups from UDP-sugars will be elucidated with the enzymes glycogen synthetase and lactose synthetase. The transfer of sugar residues from UDP-sugars are known to involve both the retention and inversion of stereochemistry at the anomeric carbon. The hypothesis to be tested is whether this group of enzymes utilizes the same mechanism for both types of transformations (inversion and retention) and whether the formation of an oxocarbonium ion is involved as an intermediate. Our approach to this problem will be to synthesize oxygen-18 isotopically labeled UDP-sugar molecules that are capable of positionally scrambling the label if such an intermediate were to form.
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