Understanding Biological Hydrogen Transfer Through the Lens of Temperature Dependent Kinetic Isotope Effects.-猫眼课题宝

DOI：

10.1021/acs.accounts.8b00226

发表时间：

2018-09-18

期刊：

Accounts of chemical research

影响因子：

18.3

通讯作者：

中科院分区：

化学1区

文献类型：

作者：

Klinman JP;Offenbacher AR

来源链接：

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6258190/

氢原子转移（HAT）是许多酶促C-H裂解机制的一个显著特征。在实现HAT动力学分离的系统中，用氢同位素（如氘）选择性标记底物，可以确定本征动力学同位素效应（KIEs）。虽然KIE的大小本身提供了信息，但最终KIE的温度依赖性的大小ΔEa = Ea(D)-Ea(H)是反应坐标的关键描述符，经常被误解（甚至忽略）。正如将在本帐户中强调的那样，ΔEa是酶催化氢转移研究中出现的最可靠的参数之一。通过HAT进行的碳氢反应的动力学参数可以与经典的“过障”或“钟形隧道修正”模型相一致。然而，这两种模型都不能解释观察到的接近零ΔEa值，许多天然酶在外来或内在扰动下增加功能。相反，已经开发了一个完整的隧道模型，该模型可以解释KIE对温度依赖的总体趋势。该模型让人想起电子隧穿的马库斯类理论，并额外加入了h原子给体-受体距离（DAD）采样项，用于有效波函数重叠；后一项的作用表现在实验确定的ΔEa中。本实验室的三种酶系统阐述了HAT的不同方面：牛磺酸双加氧酶、双铜β-单加氧酶和大豆脂加氧酶（SLO）。后者为理解氢隧穿的性质提供了一个特别引人注目的系统，显示出在活性位点铁辅助因子的近端和远端疏水残基尺寸减小时，ΔEa的系统性增加。值得注意的是，最近基于内啡肽的酶-底物复合物与SLO的研究表明，ΔEa增加的突变体的DAD增加，这与“贝尔样校正”模型不一致。总的来说，越来越多的动力学和生物物理证据证实了理解HAT的多维方法，为KIE和ΔEa的大小和趋势提供了强有力的机制解释。将SLO上最近的DFT和QM/MM计算与已开发的非绝热分析结构进行了比较，从而对基态结构和反应性提供了相当深入的了解。然而，QM/MM不能轻易地再现天然酶的小ΔEa值特征。为了捕捉这些实验观察结果，未来的理论发展可能需要从更大的构象景观中解析局部底物氘敏感模式的蛋白质运动，在此过程中更深入地了解天然酶如何进化以瞬时优化其活性位点配置。

Hydrogen atom transfer (HAT) is a salient feature of many enzymatic C-H cleavage mechanisms. In systems where kinetic isolation of HAT is achieved, selective labelling of substrate with hydrogen isotopes, such as deuterium, enables the determination of intrinsic kinetic isotope effects (KIEs). While the magnitude of the KIE is itself informative, ultimately the size of the temperature dependence of the KIE, ΔEa = Ea(D)-Ea(H), serves as a critical, and often misinterpreted (or even ignored) descriptor of the reaction coordinate. As will be highlighted in this Accounts, ΔEa is one of the most robust parameters to emerge from studies of enzyme catalyzed hydrogen transfer. Kinetic parameters for C-H reactions via HAT can appear consistent with either classical “over-the-barrier” or “Bell-like tunneling correction” models. However, neither of these models is able to explain the observation of near-zero ΔEa values with many native enzymes that increase upon extrinsic or intrinsic perturbations to function. Instead, a full tunneling model has been developed that can account for the aggregate trends in the temperature dependence of the KIE. This model is reminiscent of Marcus-like theory for electron tunneling, with the additional incorporation of an H-atom donor-acceptor distance (DAD) sampling term for effective wavefunction overlap; the role of the latter term is manifested in the experimentally determined ΔEa. Three enzyme systems from this laboratory that illustrate different aspects of HAT are presented: taurine dioxygenase, the dual copper β-monooxygenases, and soybean lipoxygenase (SLO). The latter provides a particularly compelling system for understanding the properties of hydrogen tunneling, showing systematic increases in ΔEa upon reduction in the size of hydrophobic residues both proximal and distal from the active site iron cofactor. Of note, recent ENDOR-based studies of enzyme-substrate complexes with SLO indicate an increase in DAD for mutants with increased ΔEa, observations that are inconsistent with “Bell-like correction” models. Overall, the surmounting kinetic and biophysical evidence corroborates a multi-dimensional approach for understanding HAT, offering a robust mechanistic explanation for the magnitude and trends of the KIE and ΔEa. Recent DFT and QM/MM computations on SLO are compared to the developed nonadiabatic analytical constructs, providing considerable insight into ground state structures and reactivity. However, QM/MM is unable to readily reproduce the small ΔEa values characteristic of native enzymes. Future theoretical developments to capture these experimental observations may necessitate a parsing of protein motions for local, substrate deuteration-sensitive modes from the larger conformational landscape, in the process providing deeper understanding of how native enzymes have evolved to transiently optimize their active site configurations.