Temporal and spatial resolution of distal protein motions that activate hydrogen tunneling in soybean lipoxygenase.
Temporal and spatial resolution of distal protein motions that activate hydrogen tunneling in soybean lipoxygenase.
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远端蛋白质运动的时间和空间分辨率激活大豆脂氧酶中的氢隧穿。
DOI：
10.1073/pnas.2211630120
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发表时间：
2023-03-07
影响因子：
11.1
通讯作者：
Klinman, Judith P.
中科院分区：
文献类型：
--
作者：
Zaragoza, Jan Paulo T.;Offenbacher, Adam R.;Hu, Shenshen;Gee, Christine L.;Firestein, Zachary M.;Minnetian, Natalie;Deng, Zhenyu;Fan, Flora;Iavarone, Anthony T.;Klinman, Judith P.
关键词：
使用各种生物物理工具，我们提出了一个合作的蛋白质重组的证据，起源于一个定义的蛋白质-水界面，以提供热激活驱动的活性位点氢隧道在大豆脂肪氧合酶（SLO）。当底物亚油酸被建模为SLO的可用X射线结构时，对蛋白质支架内的经典运动如何控制活性位点量子特性的原子理解变得显而易见。酶催化的动力学模型，结合分布式构象合奏与特定位点的热引发的化学反应。大豆脂肪氧合酶（SLO）为氢转移催化中的深隧穿机制提供了原型。这项工作结合了室温X射线研究与扩展的氢-氘交换实验，以定义一个催化连接的，辐射锥的脂肪族侧链连接的活性位点铁中心的SLO的蛋白质-溶剂界面。采用八种变体的SLO已附加与荧光探针在确定的表面环，纳秒荧光斯托克斯位移已被测量。我们报告了一个显着的身份的活化能（Ea）的斯托克斯位移衰减率和毫秒C-H键裂解步骤，仅限于侧链突变体在一个确定的热网络。这些发现暗示了暴露的荧光探针周围的远端蛋白质运动与控制催化的活性位点运动的直接耦合。虽然在酶功能的动态作用主要归因于一个分布式的蛋白质构象景观，所提出的数据牵连热启动，合作的蛋白质重组，发生在一个时间尺度上快于纳秒，并代表了SLO反应的生物屏障。
Using a variety of biophysical tools, we present evidence of a cooperative protein restructuring that originates at a defined protein–water interface to provide the thermal activation driving the active site hydrogen tunneling in soybean lipoxygenase (SLO). An atomistic understanding of how classical motions within the protein scaffold may control the active site quantum properties becomes apparent when the substrate linoleic acid is modeled into available X-ray structures for SLO. A dynamical model of enzyme catalysis is presented that combines distributed conformational ensembles with site-specific thermal initiation of the chemical reaction. The enzyme soybean lipoxygenase (SLO) provides a prototype for deep tunneling mechanisms in hydrogen transfer catalysis. This work combines room temperature X-ray studies with extended hydrogen–deuterium exchange experiments to define a catalytically-linked, radiating cone of aliphatic side chains that connects an active site iron center of SLO to the protein–solvent interface. Employing eight variants of SLO that have been appended with a fluorescent probe at the identified surface loop, nanosecond fluorescence Stokes shifts have been measured. We report a remarkable identity of the energies of activation (Ea) for the Stokes shifts decay rates and the millisecond C–H bond cleavage step that is restricted to side chain mutants within an identified thermal network. These findings implicate a direct coupling of distal protein motions surrounding the exposed fluorescent probe to active site motions controlling catalysis. While the role of dynamics in enzyme function has been predominantly attributed to a distributed protein conformational landscape, the presented data implicate a thermally initiated, cooperative protein reorganization that occurs on a timescale faster than nanosecond and represents the enthalpic barrier to the reaction of SLO.
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影响因子：
64.8
作者：
Eisenmesser, EZ;Millet, O;Kern, D
通讯作者：
Kern, D
影响因子：
3.3
作者：
Davarifar, Ardy;Antoniou, Dimitri;Schwartz, Steven D.
通讯作者：
Schwartz, Steven D.
影响因子：
15
作者：
Hatcher, E;Soudackov, AV;Hammes-Schiffer, S
通讯作者：
Hammes-Schiffer, S
DOI：
10.1073/pnas.1818744116
发表时间：
2019-04-02
影响因子：
11.1
作者：
Basak, Sujit;Nobrega, R. Paul;Matthews, C. Robert
通讯作者：
Matthews, C. Robert
影响因子：
2.9
作者：
Guha, S;Sahu, K;Bhattacharyya, K
通讯作者：
Bhattacharyya, K