Department of Physics, Emory University, Atlanta, Georgia.
Department of Physics, Emory University, Atlanta, Georgia.
Biophys J. 2023 Oct 3;122(19):3976-3985. doi: 10.1016/j.bpj.2023.08.020. Epub 2023 Aug 28.
We address the contribution of select classes of solvent-coupled configurational fluctuations to the complex choreography involved in configurational and chemical steps in an enzyme by comparing native and nonnative reactions conducted at different protein internal sites. The low temperature, first-order kinetics of covalent bond rearrangement of the cryotrapped substrate radical in coenzyme B-dependent ethanolamine ammonia-lyase (EAL) from Salmonella enterica display a kink, or increase in slope, of the Arrhenius plot with decreasing temperature. The event is associated with quenching of a select class of reaction-actuating collective fluctuations in the protein hydration layer. For comparison, a nonnative, radical reaction of the protein interior cysteine sulfhydryl group with hydrogen peroxide (HO) is introduced by cryotrapping EAL in an aqueous HO eutectic system. The low-temperature aqueous HO protein hydration and mesodomain solvent phases surrounding cryotrapped EAL are characterized by using TEMPOL spin probe electron paramagnetic resonance spectroscopy, including a freezing transition of the eutectic phase that orders the protein hydration layer. Kinetics of the cysteine-HO reaction in the EAL protein interior are monitored by DEPMPO spin trapping of hydroxyl radical product. In contrast to the native reaction, the linear Arrhenius relation for the nonnative cysteine-HO reaction is maintained through the solvent-protein ordering transition. The nonnative reaction is coupled to the generic local, incremental fluctuations that are intrinsic to globular proteins. The comparative approach supports the proposal that select coupled solvent-protein configurational fluctuations actuate the native reaction, and suggests that select dynamical coupling contributes to the degree of catalysis in enzymes.
我们通过比较在不同蛋白质内部位置进行的天然和非天然反应，来研究溶剂耦合构象波动对酶中构象和化学步骤所涉及的复杂协同作用的贡献。在嗜热菌属乙醇胺氨裂解酶（EAL）中，辅酶 B 依赖性的 cryotrapped 底物自由基的共价键重排的低温一级反应动力学显示出阿仑尼乌斯图的斜率增加，即随着温度的降低而增加。该事件与蛋白质水合层中特定类别的反应驱动集体波动的猝灭有关。为了进行比较，通过在水合过氧化物（HO）共晶系统中 cryotrapping EAL，引入了蛋白质内部半胱氨酸巯基与 HO 的非天然自由基反应。使用 TEMPOL 自旋探针电子顺磁共振波谱法对周围 cryotrapped EAL 的低温水合 HO 蛋白质水合和介观溶剂相进行了表征，包括共晶相的冻结转变，该转变对蛋白质水合层进行了有序化。通过 DEPMPO 自旋捕获羟基自由基产物来监测 EAL 蛋白质内部的半胱氨酸-HO 反应动力学。与天然反应相比，非天然半胱氨酸-HO 反应的线性阿仑尼乌斯关系在溶剂-蛋白质有序化转变过程中得以维持。非天然反应与球蛋白固有局部增量波动相关联。这种比较方法支持了这样一种观点，即选择耦合的溶剂-蛋白质构象波动可以驱动天然反应，并表明选择动力学耦合有助于酶的催化程度。
