Linking Protein Dynamics to Hydrogen Tunneling

蛋白质运动和催化效率之间的关系是酶巨大催化能力中最不为人所知的方面之一。我们描述和预测这种关系的能力对新的蛋白质催化剂的重新设计具有广泛的意义。该项目重点研究了一个高度相关的催化酒精氧化的酶家族（酒精脱氢酶，ADHs），其功能范围为5C（嗜冷ADH）， 25C（中温ADH）和70C（嗜热ADH）。在每种情况下，ADH催化一个氢和两个电子（氢化物离子）从底物到辅因子的转移。先前的研究表明，首先，这些反应伴随着氢化物离子的隧穿而发生；其次，氢隧穿必然与蛋白质内部的动力学特征有关。比较不同温度环境下的蛋白质，以及不同的内在动力学和稳定性，为研究蛋白质运动和催化提供了有力的实验依据。该项目将结合一系列动力学和光谱实验，使P.I.能够分别检查氢转移和蛋白质动力学特征的特性。氢转移的动力学探针将包括速率和动力学同位素效应的温度依赖性，以及氢的三种同位素（protium，氘和氚）之间的这种行为的关系。蛋白质动力学的光谱探针将包括使用氢/氘交换，连接到质谱，蛋白质荧光和核磁共振。更广泛的影响：这项工作处于酶学的前沿，将提供与新催化剂从头设计直接相关的信息。该项目正在培养一批处于职业生涯不同阶段的青年干部。该项目将包括对科研人员的培训。本科生和研究生将有机会进行研究。此外，博士后助理和初级教员的指导是本研究的一个组成部分。

The relationship between protein motions and catalytic efficiency is one of the least understood aspects of the enormous catalytic power of enzymes. Our ability to describe and predict this relationship has wide ranging implications for the de novo design of new protein catalysts. This project is focused on a highly related family of enzymes that catalyze alcohol oxidation (alcohol dehydrogenases, ADHs) that function in the range of 5C (psychrophilic ADH), 25C (mesophilic ADH) and 70C (thermophilic ADH). In each case, the ADH catalyzes the transfer of one hydrogen plus two electrons (hydride ion) from substrate to cofactor. Previous work has shown first, that these reactions occur with substantial tunneling of the hydride ion and second, that hydrogen tunneling must be linked to dynamical features within the protein. The comparison of proteins that function in different temperature niches, with different inherent dynamical and stability properties, provides a powerful experimental handle in the efforts to link protein motions and catalysis. The project will combine a range of kinetic and spectroscopic experiments that allow the P.I. to examine the properties of hydrogen transfer and protein dynamical features, respectively. Kinetic probes of hydrogen transfer will include the temperature dependence of rate and kinetic isotope effects as well as the relationship of this behavior among the three isotopes of hydrogen (protium, deuterium and tritium). Spectroscopic probes of protein dynamics will include the use of hydrogen/deuterium exchange, linked to mass spectrometry, protein fluorescence and nuclear magnetic resonance.Broader Impacts: This work is at the cutting edge of enzymology and will provide information that is directly relevant to the de novo design of new catalysts. The project is training a cadre of young personnel at different stages of careers. The project will involve training of the scientific workforce. There will be opportunities for undergraduate and graduate student research. In addition, mentoring of postdoctoral associates and beginning faculty members is an integral part of this research.