Mechanisms of Dihydroorotate Dehydrogenases

二氢乳清酸脱氢酶的机制

• 批准号: 6621399
• 负责人: BRUCE A PALFEY
• 金额: $ 20.81万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-01-01 至 2006-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6621399
• 关键词: Raman spectrometry; acidity /alkalinity; active sites; anaerobiosis; chemical chain length; chemical kinetics; computer simulation; drug design /synthesis /production; enzyme mechanism; enzyme model; enzyme substrate complex; flavin mononucleotide; intermolecular interaction; isozymes; microcalorimetry; model design /development; molecular dynamics; molecular genetics; orotate; oxidation reduction reaction; oxidoreductase; protein structure function; quinones; site directed mutagenesis; thermodynamics; thiols

DESCRIPTION: (provided by applicant) Reaction mechanisms will be determined for the family 1A dihydroorotate dehydrogenase (DHOD) from Lactococcus lactis and Enterococcus faecalis, and the family 2 enzymes from Homo sapiens and Escherichia coli. DHOD, the only redox enzyme in pyrimidine biosynthesis, is an attractive target for drug design in the treatment of many diseases, including malaria, arthritis, and Pneumocystis infections in AIDS patients. The mechanism of reduction of the enzyme-bound FMN by dihydroorotate for the two enzymes will be determined under anaerobic conditions through stopped-flow kinetic analyses, including the use of single- and double deuterium substrate isotope effects, solvent isotope effects, and the pH dependence of the rate constants. Mutant L. lactis A and E. coli enzymes will be studied in order to determine the roles that active site residues have in the reaction. The mechanism of oxidation of reduced L. lactis DHOD A by fumarate will be elucidated in stopped-flow experiments that will determine the pH dependence of the reaction, and in double-mixing experiments, the deuterium isotope effects for the transfer of each hydrogen to fumarate. The mechanism(s) of oxidation by quinones of DHOD A and the E. coli enzyme will be determined in stopped-flow experiments utilizing a range of quinone substrates. The ubiquinone isoprenyl chain-length preference of the E. coli enzyme will be determined. Enzyme-ligand interactions will be probed with Raman spectroscopy.

描述：(由申请人提供)将确定以下反应机制 乳酸乳球菌1A家族二氢罗酸脱氢酶(DHOD)和 粪肠球菌，以及智人和人类的家族2酶 大肠埃希菌。DHOD是嘧啶生物合成中唯一的氧化还原酶，是一种 在许多疾病的治疗中，药物设计具有吸引力的目标，包括 艾滋病患者的疟疾、关节炎和肺孢子虫感染。这一机制 二氢旋转酸根还原这两种酶结合的FMN将 在厌氧条件下通过停流动力学分析测定， 包括使用单、双氚衬底的同位素效应， 溶剂同位素效应，以及反应速率常数的pH依赖性。突变体L. 将对乳酸菌A和大肠杆菌酶进行研究，以确定其作用 活性中心残基在反应中的作用。氧化反应机理的研究 富马酸对乳酸乳杆菌DHOD A的抑制作用将在停流中阐明 将确定反应的pH依赖性的实验，以及在 通过二次混合实验，研究了氚同位素对转移的影响 每一个氢气都会产生富马酸根。DHOD A的苯二酚氧化机理(S) 而大肠杆菌酶将在停流实验中用 一系列的苯二酚底物。泛醌异戊二烯的链长偏好性 将测定大肠杆菌酶的活性。酶-配体的相互作用将是 用拉曼光谱进行了探测。