title: Theoretical study of enzymatic catalysis explains why the trapped covalent intermediate in the E303C mutant of glycosyltransferase GTB was not detected in the wild-type enzyme.
abstract: Hybrid quantum mechanics/molecular mechanics calculations were used to study the catalytic mechanism of the retaining human α-(1,3)-galactosyltransferase (GTBWT) and its E303C mutant (GTBE303C). Both backside (via covalent glycosyl-enzyme intermediate, CGEI) and frontside SNi-like mechanisms (via oxocarbenium-ion intermediate, OCII) were investigated. The calculations suggest that both mechanisms are feasible in the enzymatic catalysis. The nucleophilic attack of the acceptor substrate to the anomeric carbon of OCII is the rate-determining step with an overall reaction barrier (ΔE(‡) = 19.5 kcal mol(-1)) in agreement with an experimental rate constant (kcat = 5.1 s(-1)). A calculated α-secondary kinetic isotope effect (α-KIE) of 1.27 (GTBWT) and 1.26 (GTBE303C) predicts dissociative character of the transition state in agreement with experimentally measured α-KIE of other retaining glycosyltransferases. Remarkably, stable CGEI in GTBE303C compared with its counterpart in GTBWT may explain why the CGEI has been detected by mass spectrometry only in GTBE303C ( Soya N, Fang Y, Palcic MM, Klassen JS. 2011. Trapping and characterization of covalent intermediates of mutant retaining glycosyltransferases. Glycobiology, 21: 547-552).
authors.affiliation: Slovak Academy of Sciences, Institute of Chemistry, Center for Glycomics, Bratislava 845 38, Slovakia Faculty of Natural Sciences, Department of Physical and Theoretical Chemistry, Comenius University, Bratislava 4 842 15, Slovakia.
authors.forename: Adela
authors.initials: A
authors.lastname: Bobovská
authors.affiliation: Slovak Academy of Sciences, Institute of Chemistry, Center for Glycomics, Bratislava 845 38, Slovakia Department of Chemistry, Faculty of Natural Sciences, Constantine The Philosopher University, Nitra SK-949 74, Slovakia.
authors.forename: Igor
authors.initials: I
authors.lastname: Tvaroška
authors.affiliation: Slovak Academy of Sciences, Institute of Chemistry, Center for Glycomics, Bratislava 845 38, Slovakia chemkona@savba.sk.
authors.forename: Juraj
authors.initials: J
authors.lastname: Kóňa
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D055162:Biocatalysis; D003545:Cysteine / Q000737:chemistry*; D005700:Galactosyltransferases / Q000737:chemistry* / Q000235:genetics; D018698:Glutamic Acid / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D008956:Models, Chemical; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011789:Quantum Theory; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D018698:Glutamic Acid; D005700:Galactosyltransferases; C047006:N-acetyllactosaminide alpha-1,3-galactosyltransferase; D003545:Cysteine
keywords: glycosyl-enzyme intermediate; kinetic isotope effect; mechanism of glycosyltransferases; molecular modeling; structure of transition state