title: Use of an altered sugar-nucleotide to unmask the transition state for alpha(2-->6) sialyltransferase.
abstract: Rat liver alpha(2-->6) sialyltransferase catalyzes the formation of a glycosidic bond between N-acetylneuraminic acid and the 6-hydroxyl group of a galactose residue at the nonreducing terminus of an oligosaccharide. This reaction has been investigated through the use of the novel sugar-nucleotide donor substrate UMP-NeuAc. A series of UMP-NeuAc radioisotopomers were prepared by chemical deamination of the corresponding CMP-NeuAc precursors. Kinetic isotope effects (KIEs) on V/K were measured using mixtures of radiolabeled UMP-NeuAc's as the donor substrate and N-acetyllactosamine as the acceptor. The secondary beta-(2)H KIE was 1.218 +/- 0.010, and the primary (14)C KIE was 1.030 +/- 0.010. A large inverse (3)H binding isotope effect of 0.944 +/- 0.010 was measured at the terminal carbon of the NeuAc glycerol side chain. These KIEs observed using UMP-NeuAc are much larger than those previously measured with CMP-NeuAc [Bruner, M., and Horenstein, B. A. (1998) Biochemistry 37, 289-297]. Solvent deuterium isotope effects of 1.3 and 2.6 on V/K and V(max) were observed with CMP-NeuAc as the donor, and it is revealing that these isotope effects vanished with use of the slow donor substrate UMP-NeuAc. Bell-shaped pH versus rate profiles were observed for V(max) (pK(a) values = 5.5, 9.0) and V/K(UMP)(-)(NeuAc) (pK(a)values = 6.2, 9.0). The results are considered in terms of a mechanism involving an isotopically sensitive conformational change which is independent of the glycosyl transfer step. The isotope effects reveal that the enzyme-bound transition state bears considerable charge on the N-acetylneuraminic acid residue, and this and other features of this mechanism provide new directions for sialyltransferase inhibitor design.
authors.affiliation: Department of Chemistry, University of Florida, Gainesville, Florida 32611, USA.
authors.forename: M
authors.initials: M
authors.lastname: Bruner
authors.forename: B A
authors.initials: BA
authors.lastname: Horenstein
mesh_terms: D000818:Animals; D003903:Deuterium; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D019158:N-Acetylneuraminic Acid / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry*; D051381:Rats; D012799:Sialyltransferases / Q000737:chemistry*; D012997:Solvents; D013379:Substrate Specificity; D014542:Uridine Monophosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry*; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
chemical_list: D012997:Solvents; C407762:uridine monophosphate N-acetylneuraminic acid; D003903:Deuterium; D014542:Uridine Monophosphate; D012799:Sialyltransferases; D019158:N-Acetylneuraminic Acid; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase