Protein Dynamics in Catalysis by LDH and DHFR

本研究的中心目的是了解以 NAD/NADP 作为辅因子、以乳酸脱氢酶 (LDH) 和二氢叶酸还原酶 (DHFR) 作为模型系统的酶家族中氢化物转移的动力学性质。众所周知，这两种蛋白质的动力学特征对于功能很重要。这两种蛋白质是用于比较的重要模型系统，因为与（大肠杆菌）DHFR 相比，LDH 是一种更“刚性”的蛋白质，并且出于实验原因。我们的研究方法是 T 跃迁弛豫光谱，利用紫外/可见光吸收和荧光发射以及中红外吸收来跟踪从皮秒到几分钟（或更长时间）（大约 15 个十年）的结构变化。这些探针提供了实质性的结构特异性，初步研究显示了以前从未观察到的动力学特征 任何酶系统。这些研究旨在探讨酶化学在整个皮秒分钟时间范围内底物-产物内部转化的动力学和结构变化，并表征快速氢化物和质子转移步骤、循环运动、调节静电催化的运动、结合底物和活性位点残基之间的相对原子运动，以及蛋白质对活性位点结构和动力学的影响 远离活动部位的运动。测试了催化活性和非活性蛋白质亚态之间的蛋白质亚态相互转化动力学的作用以及“促进振动”对酶催化的作用。我们将探索和表征模拟米氏复合物的蛋白质复合物的动力学，模拟过渡态某些方面的复合物，以及生产性酶/底物<->酶/产物内​​部转换。此外，还将研究关键蛋白质突变体对动力学的影响，以便将动力学与催化联系起来。

The central aim of this study is to understand the dynamical nature of hydride transfer in the family of enzymes that use NAD/NADP as cofactors with lactate dehydrogenase (LDH) and dihydrofolate reductase (DHFR) as model systems. It is known that dynamical features of these two proteins are important for function. The two proteins are important model systems for comparison since LDH is a more 'rigid' protein compared to (E. coli) DHFR and for experimental reasons. The approach for our study is T-jump relaxation spectroscopy employing UV/vis absorption and fluorescence emission and mid-IR absorption to follow changes in structure from ps to minutes (or longer), some 15 decades of time. These probes provide substantial structural specificity, and preliminary studies exhibit dynamical features never before observed on any enzymic system. The studies are designed to probe the kinetics and structural changes of substrate-product inner conversion of on-enzyme chemistry over the entire ps-minutes time range and characterize fast hydride and proton transfer steps, loop motion, motions that modulate electrostatic catalysis, relative atomic motion between the bound substrate and active site residues, and the effects on the structure and dynamics at the active site by protein motions far from the active site. The role of interconversion kinetics of protein sub-states between catalytically active and inactive protein sub-states and 'promoting vibrations' on on-enzyme catalysis are tested. We shall probe and characterize the dynamics of protein complexes that mimic Michaelis complexes, complexes that mimic some aspects of the transition state, as well as productive enzyme/substrate <-> enzyme/product inner conversion. In addition, the effects of key protein mutants on the dynamics will be studied in order to relate dynamics to catalysis.