Extremely Elevated Room-Temperature Kinetic Isotope Effects Quantify the Critical Role of Barrier Width in Enzymatic C鈥揌 Activation

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• 作者：Shenshen Hu ; Sudhir C. Sharma ; Alexander D. Scouras ; Alexander V. Soudackov ; Cody A. Marcus Carr ; Sharon Hammes-Schiffer ; Tom Alber ; Judith P. Klinman
• 刊名：Journal of the American Chemical Society
• 出版年：2014
• 出版时间：June 11, 2014
• 年：2014
• 卷：136
• 期：23
• 页码：8157-8160
• 全文大小：299K
• 年卷期：v.136,no.23(June 11, 2014)
• ISSN：1520-5126

文摘

The enzyme soybean lipoxygenase (SLO) has served as a prototype for hydrogen-tunneling reactions, as a result of its unusual kinetic isotope effects (KIEs) and their temperature dependencies. Using a synergy of kinetic, structural, and theoretical studies, we show how the interplay between donor鈥揳cceptor distance and active-site flexibility leads to catalytic behavior previously predicted by quantum tunneling theory. Modification of the size of two hydrophobic residues by site-specific mutagenesis in SLO reduces the reaction rate 10⁴-fold and is accompanied by an enormous and unprecedented room-temperature KIE. Fitting of the kinetic data to a non-adiabatic model implicates an expansion of the active site that cannot be compensated by donor鈥揳cceptor distance sampling. A 1.7 脜 resolution X-ray structure of the double mutant further indicates an unaltered backbone conformation, almost identical side-chain conformations, and a significantly enlarged active-site cavity. These findings show the compelling property of room-temperature hydrogen tunneling within a biological context and demonstrate the very high sensitivity of such tunneling to barrier width.