STRUCTURE AND FUNCTION OF ALLYLIC REARRANGEMENT ENZYMES

烯丙基重排酶的结构和功能

• 批准号: 3289949
• 负责人: John M. Schwab
• 金额: $ 17.49万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-08-01 至 1994-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3289949
• 关键词: X ray crystallography; adduct; allyl compound; chemical hydration; drug design /synthesis /production; enzyme induction /repression; enzyme model; enzyme structure; hydrolase; molecular rearrangement; nuclear magnetic resonance spectroscopy; stereochemistry

Further studies on enzymes catalyzing allylic rearrangments are proposed. Beta-Hydroxydecanoyl thiol ester dehydrase (E. coli), which equilibrates thiol esters of (R)-3hydroxydecanoic acid, E-2-decenoic acid, and Z-3-decenoic acid, is the key enzyme in the biosynthesis of bacterial unsaturated fatty acids. Dehydrase is rapidly inactivated by the mechanism-based inactivator 3decynoyl-NAC (NAC =N-acetylcysteamine thiol ester), via dehydrase-catalyzed isomerization to 2,3decadienoyl-NAC. The amino acid sequence of dehydrase has recently been determined, and the residue that is modified by 3-decynoyl-NAC (and is therefore implicated as the active site base) was shown to be His-70. The long-term objective is to further knowledge of enzymw structure and function. Such knowledge is critical for the rational design of drup that act at the enzyme level. The specific aims of this proposal include (a) determination (by 15N NMR) of which of dehydrase's imidazole nitrogens is modified by 3-decynoyl-NAC; (b) synthesis and application of novel enzyme inactivators, with analysis of protein by peptide mapping and multinuclear NMR, in order to identify catalytically important residues other than His-70; (c) synthesis and evaluation of ketone analogs of dehydrase's thiol ester substrates as potential nonhydrolyzable substrates and inhibitors; (d) determinnation of the three-dimensional crystal structures of dehydrase that has been inactivated with novel 3-decynoic acid thiol esters, including the ACP thiol ester; (e) complete characterization of dehydrase by multinuclear NMR, allowing comparison of crystal- and solution-state samples and enabling the identification of dehydrase-ACP contact points; (f) generation of mutant dehydrases, with modified active site residues and modified acyl chain binding regions, to test the roles of amino acid residues that are suspected to be important in catalysis and to generate E. coli strains with altered unsaturated fatty acids; (g) reevaluation of the stereochemical course of the allylic rearrangement catalyzed by the Betahydroxydodecanoyl thiol ester dehydrase component of Brevibacterium ammoniagenes fatty acid synthetase (by 2H-decoupled 1H,13C chemical shift correlation spectroscopy), in order to test for mechanistic unifomity among enzymes catalyzing allylic rearrangements.

对催化烯丙位还原的酶作了进一步的研究。 β-羟基癸酰基硫羟酸酯酶（E.大肠杆菌）， （R）-3羟基癸酸、E-2-癸烯酸和 Z-3-癸烯酸是细菌生物合成的关键酶 不饱和脂肪酸脱水酶迅速失活， 基于机理的灭活剂3-癸炔酰基-NAC（NAC = N-乙酰基半胱胺硫醇 酯），通过脱氢酶催化的异构化反应生成2，3-癸二烯酰基-NAC。的 最近已经确定了该酶的氨基酸序列， 被3-癸炔酰基-NAC修饰的残基（并且因此被暗示为 活性位点碱基）被证明是His-70。 长期目标是进一步了解酶的结构， 功能这些知识对于drup的合理设计至关重要， 在酶水平上起作用。这项建议的具体目标包括：（a） 确定（通过15 N NMR）其中哪一个酶的咪唑氮是 3-癸炔酰基-NAC修饰;（B）新酶的合成和应用 用肽图谱和多核分析法分析蛋白质的灭活剂 NMR，以鉴定除以下之外的催化重要的残基： His-70;（c）合成和评价乙酰氨基转移酶巯基的酮类似物 酯底物作为潜在的不可水解底物和抑制剂; (d)胰蛋白酶三维晶体结构的测定 其已经用新的3-癸炔酸硫醇酯灭活， 包括ACP硫羟酸酯;（e）完整表征 通过多核NMR，可以比较晶体和溶液状态 样品，并能够识别酶-ACP接触点; (f)产生具有经修饰的活性位点残基的突变体酶， 修饰的酰基链结合区，以测试氨基酸 被怀疑是重要的催化和产生的残留物 E.不饱和脂肪酸改变的大肠杆菌菌株;（g）重新评估 催化烯丙基重排的立体化学过程 短杆菌的β-羟基十二烷酰基硫酯酶组分 产氨脂肪酸合成酶（2 H-去偶1H，13 C化学位移 相关光谱），以测试 催化烯丙基重排的酶。