The effect of electrostatic shielding on H tunneling in R67 dihydrofolate reductase.
The effect of electrostatic shielding on H tunneling in R67 dihydrofolate reductase.
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静电屏蔽对 R67 二氢叶酸还原酶中 H 隧道的影响。
DOI：
10.1002/cbic.200900451
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发表时间：
2009
期刊：
影响因子：
--
通讯作者：
Kohen,Amnon
中科院分区：
文献类型：
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作者：
Yahashiri,Atsushi;Nimrod,Guy;Ben-Tal,Nir;Howell,ElizabethE;Kohen,Amnon
二氢叶酸还原酶（DHFR）催化NADPH和二氢叶酸之间的氢化物（H）转移反应，并产生四氢叶酸和NADP+。R67 DHFR是质粒编码的酶，并且由于其基因组、结构和动力学特性而被认为是"原始酶"。[1，2]有趣的是，R67 DHFR的动力学研究显示，随着离子强度的增加，H-转移速率增加。[3]为了评估这种速率增强的来源，测量了内在动力学同位素效应（KIE）的温度依赖性，并在低和高离子强度下评估了H-转移步骤的性质。在高离子强度下，KIE对温度的依赖性小于在较低离子强度下。这些研究结果进行了评估，使用马库斯样模型，这表明，在较高的离子强度，氢化物的供体和受体更好地定向H-隧穿比相同的系统在较低的离子强度。该比较解决了使反应坐标进入隧道就绪构象的系统制备水平。虽然影响很小，但具有统计学显著性，如补充信息（SI-表S2）中提供的比较数据和标准差所示。这些数据证明了为研究该系统而开发的方法的高灵敏度（参见SI中的详细方法）。计算了低离子强度和高离子强度下静电势面的差异，理论结果为实验数据增加了分子视角。动力学同位素效应（KIE）是指两种反应物的速率之比，这两种反应物的同位素组成不同。本征KIE的温度依赖性对反应的势面和动力学敏感。它作为一个很好的探针量子力学H-隧道和组织的H-供体和受体在酶的活性位点。[4，5]评估固有KIE的一种方法涉及测量氢的三种同位素的KIE。[5-7]这种方法可以评估的H转移反应的性质，在酶催化的酶反应的复杂的动力学级联的化学步骤，特别关注。由于氢的三种不同同位素1H（H）、2H（D）和3H（T）的质量比很大，测量结果中大而明显的KIE提供了反应势面的宝贵信息。[8]比如说，
Dihydrofolate reductase (DHFR) catalyzes the hydride (H) transfer reaction between NADPH and dihydrofolate, and produces tetrahydrofolate and NADP+. R67 DHFR is a plasmid encoded enzyme, and is considered a “primitive enzyme” due to its genomic, structural, and kinetic properties.[1, 2] Interestingly, kinetic studies of R67 DHFR show an enhancement in H-transfer rate with increasing ionic strength.[3] To evaluate the source of this rate enhancement, the temperature dependency of intrinsic kinetic isotope effects (KIEs) was measured and the nature of the H-transfer step was evaluated at low and high ionic strengths. At high ionic strength, the KIEs were less temperature dependent than at lower ionic strength. These findings were evaluated using a Marcus-like model, which suggests that at higher ionic strength, the donor and acceptor of the hydride were better oriented for H-tunneling than the same system at lower ionic strength. This comparison addresses the level of system preparation that brings the reaction coordinate into a tunneling-ready conformation. While the effect is small, it is statistically significant, as apparent from the comparative data and standard deviations presented in the Supplementary Information (SI–Table S2). These data demonstrate the high sensitivity of the methodology that was developed to study this system (see detailed methods in the SI). The differences in electrostatic potential surface between low and high ionic strengths were calculated, and the theoretical findings add a molecular perspective to the experimental data.A kinetic isotope effect (KIE) is the ratio of rates of two reactants that only differ in their isotopic composition. The temperature dependence of intrinsic KIEs is sensitive to a reaction’s potential surfaces and dynamics. It serves as an excellent probe for quantum mechanical H-tunneling and the organization of the H-donor and acceptor at the enzyme’s active site.[4, 5] One way to assess the intrinsic KIEs involves measurements of KIEs for the three isotopes of hydrogen.[5–7] This methodology may evaluate the nature of the H transfer reaction in enzyme catalysis by specifically focusing on the chemical step in the complex kinetic cascade of an enzymatic reaction. Because of the large mass ratio of three different isotopes of hydrogen, 1H (H), 2H (D) and 3H (T), large and distinct KIEs on the measurements give precious information on the reaction potential surface.[8] For instance,