| Sialyltransferases are glycosyltransferases that catalyze the formation of a glycosidic bond to NeuAc (sialic aid). The resulting sialylated glycoproteins and glycolipids are frequently involved in cell-cell and cell-pathogen binding interactions in addition to other biologically important phenomena. The chemical and catalytic mechanisms of glycosyltransferases have not been well characterized, and this includes sialyltransferases. This dissertation describes mechanistic studies of sialyltransferases, with the long term goal being to use this information for logical inhibitor design.;The first part of this work is a record of the synthesis of the compounds necessary to complete the kinetic experiments. The natural donor substrate for this system, cytidine-5'-monophosphate neuraminic acid (CMP-NeuAc), has been synthesized with a wide variety of isotopic labels located in specific positions. A novel unnatural "slow" donor substrate uridine-5'-monophosphate neuraminic acid (UMP-NeuAc) has also been synthesized containing these isotopic substitutions. The approaches to synthesize, purify, and characterize these molecules are described.;The use of the above mentioned substrates in a variety of kinetic experiments with rat liver alpha(2→6) sialyltransferase is discussed next. These experiments include Lineweaver-Burk analysis, pH-rate profiles, isotope trapping, and inhibitor analysis. The results of these experiments are discussed. Additionally, the results of similar experiments completed using the two different donor substrates (CMP-NeuAc and UMP-NeuAc) are presented.;A variety of kinetic isotope effects have been measured on this system. These effects include beta-dideuterium, primary 14C, secondary 14C, 18O leaving group, and solvent isotope effects. These have been measured for the enzyme catalyzed reaction, as well as for the acid catalyzed hydrolysis reactions of the substrates which serves as a model for the enzyme catalyzed reaction. Comparisons are made of the isotope effects measured using the two different donor substrates (CMP-NeuAc and UMP-NeuAc).;The dissertation is concluded with the KlEs measured for another member of the sialyltransferase family, rat liver alpha(2→3) sialyltransferase. The KIE data for the rat liver alpha(2→6) and alpha(2→3) sialyltransferases allow for a comparison of the transition-states of these two enzymes. |
