Protein Dynamics in Catalysis by LDH and DHFR

本研究的中心目的是了解以乳酸脱氢酶（LDH）和二氢叶酸还原酶（DHFR）为模型系统，以NAD/NADP为辅助因子的酶家族中氢化物转移的动力学性质。众所周知，这两种蛋白的动力学特性对其功能至关重要。这两种蛋白质是比较的重要模型系统，因为LDH与（大肠杆菌）DHFR相比是一种更“刚性”的蛋白质，并且出于实验原因。我们的研究方法是t跳弛豫光谱，利用紫外/可见吸收和荧光发射和中红外吸收来跟踪从ps到几分钟（或更长时间）的结构变化，大约15年的时间。这些探针提供了大量的结构特异性，初步研究显示了以前从未观察到的动力学特征

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.