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

• Published in: Glycobiology (Oxford) 2015-01, Vol.25 (1), p.3-7
• Main Authors: Bobovská, Adela, Tvaroška, Igor, Kóňa, Juraj
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; Binding Sites; Biocatalysis; Cysteine - chemistry; Galactosyltransferases - chemistry; Galactosyltransferases - genetics; Glutamic Acid - chemistry; Humans; Kinetics; Mass Spectrometry; Models, Chemical; Mutation; Protein Binding; Protein Conformation; Quantum Theory; Substrate Specificity; Thermodynamics
• ISSN: 0959-6658; 1460-2423
• DOI: 10.1093/glycob/cwu085

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).