title: Primary 13C and beta-secondary 2H KIEs for trans-sialidase. A snapshot of nucleophilic participation during catalysis.
abstract: Trypanosoma cruzi trans-sialidase catalyzes a novel reaction that involves the transfer of sialic acid between host and parasite glycoconjugates. In this paper, we report kinetic isotope effect studies on recombinant trans-sialidase. beta-Dideuterium and primary 13C isotope effects were measured for a good substrate, sialyl-lactose, and a slow substrate, sialyl-galactose, in both acid-catalyzed solvolysis and enzymatic transfer reactions. The beta-dideuterium isotope effect for sialyl-lactose in the acid hydrolysis reaction was 1.113 +/- 0.012. The primary 13C isotope effects for hydrolysis of sialyl-lactose and sialyl-galactose were 1. 016 +/- 0.011 and 1.015 +/- 0.008, respectively. In the enzymatic transfer reactions, the beta-dideuterium and primary 13C effects for sialyl-galactose were 1.060 +/- 0.008 and 1.032 +/- 0.008, respectively. The isotope effects for hydrolysis describe a dissociative SN1-like mechanism, and these data are contrasted by the data for the enzyme-catalyzed reaction. The enzymatic deuterium isotope effects are lower by a factor of 2, but the primary carbon isotope effects are higher by a factor of 2. This pattern describes a mechanism involving nucleophilic participation in the rate-determining transition state.
authors.affiliation: Department of Chemistry, University of Florida, Gainesville, Florida 32611, USA.
authors.forename: J
authors.initials: J
authors.lastname: Yang
authors.forename: S
authors.initials: S
authors.lastname: Schenkman
authors.forename: B A
authors.initials: BA
authors.lastname: Horenstein
mesh_terms: D000818:Animals; D000953:Antigens, Protozoan / Q000737:chemistry; D002240:Carbohydrate Sequence; D002247:Carbon Isotopes; D002384:Catalysis; D003903:Deuterium / Q000737:chemistry*; D005732:Gangliosides / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry*; D007700:Kinetics; D007785:Lactose / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000737:chemistry; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000737:chemistry*; D051381:Rats; D012794:Sialic Acids / Q000138:chemical synthesis / Q000737:chemistry; D000080506:Sialyl Lewis X Antigen; D013379:Substrate Specificity; D014349:Trypanosoma cruzi / Q000201:enzymology*
chemical_list: D000953:Antigens, Protozoan; D002247:Carbon Isotopes; D005732:Gangliosides; D006023:Glycoproteins; D012794:Sialic Acids; D000080506:Sialyl Lewis X Antigen; C094688:sialyl Lewis(x) ganglioside; C000020:N-acetylneuraminoyllactose; D003903:Deuterium; C069715:trans-sialidase; D009439:Neuraminidase; D007785:Lactose