STEREOCHEMISTRY AND MECHANISMS OF ENZYME REACTIONS

催化烯丙基和丙叉基的酶的进一步研究 建议重新安排。 β-羟基十二烷基硫酸酯脱水酶(E.Coli)对 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-癸烯酸硫代酯。组氨酸残留物显然是唯一 活性部位碱/共轭酸，尽管没有确凿证据 一种必需的酪氨酸。脱水酶不可逆地被失活 底物类似物3-十一醇-NAC(NAC=N-乙酰半胱胺)，通孔 2，3-十二烯基-NAC。烯丙硫酸酯烷基化香精 组氨酸。 从猪肝转化中分离到一种丙二烯-乙炔异构酶(AAI) 2，3-和3，4-烯醇基硫代酯，尽管它的作用更有效 3-乙烯基和2，3-二烯基硫酸酯底物。AAI不受以下因素影响 2，3-十二烯基-NAC。 关于脱水酶和AAI的拟议研究包括：(A)用15N测定 核磁共振)，其中脱水酶的(不等价)咪唑氮成为 附着在灭活剂的C-3上；(B)双官能团的应用 失活剂，同时具有3，4-乙酰基官能团和第二个 反应性(或潜在性反应性)官能团，如不同的 活性部位区域；(C)脱水酶结晶和初步 X射线结晶学研究；(D)设计、合成和实施 新的潜在AAI失活剂的研究；(E)分离和鉴定 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 控制功能的功能，特别是在活动站点 生物合成所必需的、唯一的酶 厌氧条件下的不饱和脂肪酸，并在这样做时，(B) 开发将灭活剂多重连接到靶标酶上的方法； (C)得出有关活跃场地结构的有用信息，以及 AAI的作用机制，一种不寻常的，可能在代谢上很重要的 酶，其机理信息很少；(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。

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.