STEREOCHEMISTRY AND MECHANISMS OF ENZYME REACTIONS

立体化学和酶反应机制

• 批准号: 3289947
• 负责人: John M. Schwab
• 金额: $ 10.35万
• 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/3289947
• 关键词: Clostridium; chemical hydration; enzyme induction /repression; hydrolase; hydroxyl group; stereochemistry

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.

烯丙酸和炔丙酸催化酶的进一步研究 建议重新安排。 β-羟基癸硫酯脱水酶（大肠杆菌）对于 厌氧条件下不饱和脂肪酸的生物合成， 催化 (R)-3-羟基癸酸酯、E-2-癸烯酸酯和 Z-3-癸烯酸硫酯。 组氨酸残基显然是唯一的 活性位点碱/共轭酸，尽管没有确凿的证据 为必需的酪氨酸。 脱水酶不可逆地失活 底物类似物 3-癸醇-NAC（NAC = N-乙酰半胱胺），via 2,3-癸二烯酰基-NAC。 联二烯硫酯烷基化必需的 组氨酸。 从猪肝中分离出的丙二烯-乙炔异构酶（AAI）可相互转化 2,3- 和 3,4- 烯酰硫酯，尽管它的作用更有效 3-炔酰基和2,3-二烯酰基硫酯底物。 AAI 不受影响 2,3-癸二烯酰基-NAC。 关于脱水酶和 AAI 的拟议研究包括： (a) 通过 15N 测定 NMR）其中脱水酶的（非等价）咪唑氮变为 附着于灭活剂的C-3； (b) 双功能的应用 灭活剂，同时具有 3,4-乙炔羰基功能和第二个功能 反应性（或潜在反应性）官能团，作为不同的探针 活性位点区域； (c)脱水酶的结晶和初步 X射线晶体学研究； (d) 设计、综合和实施 AAI 的新型潜在灭活剂； (e) 分离和表征 来自替代来源（希望是细菌）的 AAI，以便 （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，特别是在活性位点，控制功能 酶是生物合成中独特且不可或缺的酶 不饱和脂肪酸在厌氧条件下，并且在此过程中，(b) 开发将灭活剂多次附着到目标酶上的方法； (c) 获取有关活性位点结构的有用信息以及 AAI 的作用机制，一种不寻常且可能具有重要代谢意义的物质 酶的机械信息很少； (d) 进一步 展示氟取代化合物的实用性和多功能性 作为亲和标记、“自杀”底物和竞争性酶 抑制剂； (e) 开发 AAI 的替代来源。