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.
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.
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酶催化的理论研究解释了为什么在野生型酶中没有检测到糖基转移酶 GTB E303C 突变体中捕获的共价中间体。
作者：
Adela Bobovská;I. Tvaroška;J. Kóňa
采用混合量子力学/分子力学计算研究了保留人α-(1,3)-半乳糖基转移酶(GTBWT)及其E303C突变体(GTBE303C)的催化机制。研究了背面（通过共价糖基酶中间体，CGEI）和正面 SNi 样机制（通过氧碳鎓离子中间体，OCII）。计算表明这两种机制在酶催化中都是可行的。受体底物对 OCII 异头碳的亲核攻击是速率决定步骤，其总反应势垒 (ΔE(‡) = 19.5 kcal mol(-1)) 与实验速率常数 (kcat = 5.1 s(-1)) 一致。计算得出的 α-二级动力学同位素效应 (α-KIE) 为 1.27 (GTBWT) 和 1.26 (GTBE303C)，预测过渡态的解离特征，与实验测量的其他保留糖基转移酶的 α-KIE 一致。值得注意的是，与 GTBWT 中的对应物相比，GTBE303C 中的稳定 CGEI 可以解释为什么仅在 GTBE303C 中通过质谱法检测到 CGEI（Soya N、Fang Y、Palcic MM、Klassen JS.2011.Trapping and characterization of covalent middles of mutoring angiosyltransferases.Glycobiology, 21:547-552）。
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).
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影响因子：
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作者：
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DOI：
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DOI：
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期刊：
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影响因子：
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作者：
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作者：
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