STEREOCHEMISTRY AND MECHANISMS OF ENZYME REACTIONS

立体化学和酶反应机制

• 批准号: 3289950
• 负责人: John M. Schwab
• 金额: $ 10.68万
• 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 1989-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3289950
• 关键词: Clostridium; chemical hydration; enzyme induction /repression; hydrolase; hydroxyl group; stereochemistry; tritium

Further studies of enzymes catalyzing allylic and propargylic rearrangements are proposed. Beta-Hydroxydecanolythioester dehydrase (E. coli) is crucial to the biosynthesis of unsaturated fatty acids under anaerobic conditions, catalyzing interconversion of (R)-3-hydroxydecanoate, E-2-decenoate, and Z-3-decenoate thioesters. A histidine residue is apparently the sole active site base/conjugate acid, although there is inconclusive evidence for an essential tyrosine. Dehydrase is irreversibly inactivated by the substrate analog 3-decynol-NAC (NAC = N-acetycysteamine), via 2,3-decadienoyl-NAC. The allenic thioester alkylates the essential histidine. An allene-acetylene isomerase (AAI) isolated from hog liver interconverts 2,3- and 3,4-enoyl thioesters, though it functions more efficiently with 3-ynoyl and 2,3-dienoyl thioester substrates. AAI is unaffected by 2,3-decadienoyl-NAC. Proposed studies on dehydrase and AAI include: (a) determination by 15N NMR) of which (nonequivalent) imidazole nitrogen of dehydrase becomes attached to C-3 of the inactivator; (b) application of bifunctional inactivators, bearing both a 3,4-acetylenic carbonyl function and a second reactive (or latently reactive) functional group, as a probe of a different region of the active site; (c) crystallization of dehydrase and preliminary x-ray crystallographic studies; (d) design, synthesis, and implementation of novel potential inactivators of AAI; (e) isolation and characterization of AAI from an alternative (hopefully bacterial) source, in order to (ultimately) amplify the yield of AAI and to facilitate its isolation. The significance of this project is multifold: (a) to identify structural features, particularly at the active site, that control the function of the enzyme that is unique, and indispensible, to the biosynthesis of unsaturated fatty acids under anaerobic conditions, and in doing so, (b) to develop methods for multiple attachment of inactivators to target enzymes; (c) to derive useful information regarding the active site structure and mechanism of action of AAI, an unusual and possibly metabolically important enzyme for which there is little mechanistic information; (d) to further demonstrate the utility and versatility of fluorine-substituted compounds as affinity labels, "suicide " substrates, and competitive enzyme inhibitors; (e) to develop an alternative source of AAI.

催化烯丙基和炔丙基的酶的进一步研究 建议重新安排。 β-羟基癸烷聚硫酯脱氢酶（E.大肠杆菌）对 在厌氧条件下生物合成不饱和脂肪酸， 催化（R）-3-羟基癸酸酯、E-2-癸烯酸酯和 Z-3-癸烯酸硫酯。 组氨酸残基显然是 活性位点碱/共轭酸，尽管没有确凿的证据 必需的酪氨酸 脱水酶不可逆地被 底物类似物3-癸炔-NAC（NAC = N-乙酰基半胱胺），通过 2，3-癸二烯酰基-NAC。 丙二烯硫酯使必需的 组氨酸。 猪肝中的一种丙二烯-乙炔异构酶（AAI） 2，3-和3，4-烯酰基硫酯，尽管它与 3-炔酰基和2，3-二烯酰基硫酯底物。 AAI不受 2，3-癸二烯酰基-NAC。 目前，对AAI和AAI的研究主要包括：（a）15 N NMR），其中酶的（非当量）咪唑氮变成 连接到灭活剂的C-3;（B）双官能 失活剂，带有3，4-炔属羰基官能团和第二个 反应性（或潜在反应性）官能团，作为不同的探针， 活性位点的区域;（c）结晶化酶和初步的 X射线晶体学研究;（d）设计、合成和实施 AAI的新型潜在灭活剂;（e）分离和表征 AAI从替代（希望是细菌）来源，以 （最终）放大AAI的产量并促进其分离。 该项目的意义是多方面的：（a）确定结构 特征，特别是在活性部位，控制着 酶是独特的，必不可少的，生物合成的 不饱和脂肪酸，并且在这样做时，（B） 开发灭活剂与靶酶多重连接的方法; (c)获得有关活性部位结构的有用信息， AAI的作用机制，一种不寻常的，可能在代谢上重要的 酶，其中几乎没有机械信息;（d）进一步 证明了氟取代化合物的实用性和通用性 作为亲和标记、“自杀“底物和竞争酶 （e）开发AAI的替代来源。