Protein Dynamics in Catalysis by LDH and DHFR

LDH 和 DHFR 催化中的蛋白质动力学

基本信息

批准号：
7616246
负责人：
Robert Callender
金额：
$ 26.48万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7616246
关键词：
Active Sites Adopted Affect Amides Anisotropy Binding Binding Sites Biological Models Catalysis Chemicals Chemistry Collaborations Complex Coupled Dihydrofolate Reductase Electrostatics Environment Enzymes Escherichia coli Event Exhibits Family Fluorescence Frequencies Indoles Isotopes Kinetics Label Lactate Dehydrogenase Lactate Dehydrogenases Lasers Measures Molecular Conformation Monitor Motion Mutation NAD-pyruvate adduct NADH NADP Nature Nicotinamide adenine dinucleotide Oxidation-Reduction Pathway interactions Pliability Positioning Attribute Principal Investigator Property Protein Dynamics Proteins Protons Pyruvate Pyruvates Range Rate Reaction Relative (related person)Relaxation Reporting Research Design Research Personnel Rest Role Specificity Spectrum Analysis Stretching Structure System Testing Thinking Time Variant Vertebral column absorption carboxylate cofactor design enzyme substrate indole inhibitor/antagonist lactate dehydrogenase 1 mutant programs protonation research study response tool vibration

项目摘要

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.

这项研究的核心目的是了解使用NAD/NADP作为乳酸脱氢酶（LDH）和二氢叶酸还原酶（DHFR）作为模型系统的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。这两种蛋白质是进行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。我们研究的方法是使用UV/VIS吸收和荧光发射和MID-IR吸收的T型松弛光谱，以遵循从PS到几分钟（或更长）（或更长）（大约15年的时间）结构的变化。这些探针提供了实质性的结构特异性，初步研究表现出前所未有的动力学特征任何酶系统。该研究旨在探测整个PS分钟时间范围内酶内化学内部化学内部转化的动力学和结构变化远离活动站点的运动。测试了催化活性和非活性蛋白质亚酸盐和“促进振动”对酶场催化的蛋白质子菌的相互转换动力学的作用。我们应探测和表征模仿Michaelis复合物的蛋白质复合物的动力学，模仿过渡态的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，将研究关键蛋白突变体对动力学的影响，以将动力学与催化相关。