Journal Article

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

Adela Bobovská, Igor Tvaroška and Juraj Kóňa

in Glycobiology

Volume 25, issue 1, pages 3-7
Published in print January 2015 | ISSN: 0959-6658
Published online August 2014 | e-ISSN: 1460-2423 | DOI:

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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).

Keywords: glycosyl-enzyme intermediate; kinetic isotope effect; mechanism of glycosyltransferases; molecular modeling; structure of transition state

Journal Article.  3444 words.  Illustrated.

Subjects: Carbohydrates