Effect of solvent viscosity on the activation barrier of hydrogen tunneling in the lipoxygenase reaction
Effect of solvent viscosity on the activation barrier of hydrogen tunneling in the lipoxygenase reaction
复制标题
溶剂粘度对脂氧合酶反应中氢隧道活化势垒的影响
DOI：
10.1016/j.abb.2023.109740
复制
发表时间：
2023
影响因子：
3.9
通讯作者：
Offenbacher, Adam R.
中科院分区：
文献类型：
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作者：
Guevara, Luis;Gouge, Melissa;Ohler, Amanda;Hill, S. Gage;Patel, Soham;Offenbacher, Adam R.
酶反应中的氢隧穿在将蛋白质热运动与催化的化学步骤联系起来方面发挥了重要作用。脂氧合酶（LOX）已作为此类反应的模型系统，展示了与多不饱和脂肪酸的酶促C-H键裂解相关的深氢隧穿机制。在这里，我们研究了溶剂粘度对蛋白质热运动与LOX催化使用海藻糖和葡萄糖作为粘原的影响。动力学分析的范例植物直向同源物，大豆脂肪氧合酶（SLO），与亚油酸的反应，揭示了一级速率常数，kcat，或活化能，Ea没有影响。SLO活性位点突变体显示variyingEas，这已被用来探测催化相关的运动，进一步的研究，同样没有提供证据的粘度依赖性的运动。动力学分析扩展到一个代表性的真菌LOX从mM。MoLOX和人LOX，15-LOX-2。虽然MoLOX的行为与SLO相似，但我们表明粘原抑制15-LOX-2活性。后者暗示动物LOX反应中的粘度敏感的构象运动。这些数据提供了深入了解水的水合层在促进氢（量子）在LOX隧道的作用。
Hydrogen tunneling in enzyme reactions has played an important role in linking protein thermal motions to the chemical steps of catalysis. Lipoxygenases (LOXs) have served as model systems for such reactions, showcasing deep hydrogen tunneling mechanisms associated with enzymatic C–H bond cleavage from polyunsaturated fatty acids. Here, we examined the effect of solvent viscosity on the protein thermal motions associated with LOX catalysis using trehalose and glucose as viscogens. Kinetic analysis of the reaction of the paradigm plant orthologue, soybean lipoxygenase (SLO), with linoleic acid revealed no effect on the first-order rate constants,kcat, or activation energy,Ea. Further studies of SLO active site mutants displaying varyingEas, which have been used to probe catalytically relevant motions, likewise provided no evidence for viscogen-dependent motions. Kinetic analyses were extended to a representative fungal LOX fromM. oryzae, MoLOX, and a human LOX, 15-LOX-2. WhileMoLOX behaved similarly to SLO, we show that viscogens inhibit 15-LOX-2 activity. The latter implicates viscogen sensitive, conformational motions in animal LOX reactions. The data provide insight into the role of water hydration layers in facilitating hydrogen (quantum) tunneling in LOX.