ENZYMES OF THE META FISSION PATHWAY

有几种微生物能够利用芳香烃作为唯一的 碳和能源的来源。这一能力源于高度 将芳香烃转化为 克雷布斯循环中的中间体。最初，芳香烃是 转化为邻苯二酚或取代的邻苯二酚。随后， 儿茶酚化合物是由许多SO- 被称为超裂变途径。由这些组成的酵素电池 变质裂变途径具有丰富的机械性、结构性和 进化论问题，并提出一个有吸引力的战略 环境中芳香烃的降解。 这项研究的长期目标是应用来自蛋白质的技术 化学、分子生物学和x射线结晶学 对两种酶的机制、结构和进化进行了深入的研究 元裂变途径--儿茶酚和高原儿茶酸的元裂变 小路。在这段资助期内，我们会继续研究4- 草酸互变麦芽酶(4-OT)、4-草酸脱羧酶(4-OD)、 和乙烯基丙酮酸水合酶(VPH)。所有的都在儿茶酚的裂变中 路径。 在此期间，我们的主要具体目标按优先顺序列出 资助期将是：(L)进一步检验“锚定”假说 对于4-OT使用底物类似物；(2)表征4-OT的行为 具有潜在的基于乙炔机理的抑制剂和潜在的 亲和标记；(3)检测Pro-1在4-OT机制中的作用 通过化学合成和突变体的表征；(4)继续 作为溶液结构前奏的4-OT的核磁共振研究；(5)确定 ~(13)C动力学同位素对非酶反应和4-OD反应的影响 2-氧代-3-己二酸酯的脱羧化反应及其与文献报道的结果比较 先前确定的草乙酸酯脱羧基；(6) 确定了所提出的烯丙基异构化的空间历程和a VPh催化的水合反应如果构型 可以建立中间体；(7)指定C-5的立体化学 VPH反应的产物；以及(8)检查VPH的行为 其底物的乙炔类似物。 拟议的研究将解决关键的机制和进化问题。 提出的问题似乎是一种常见的策略， 降解儿茶酚类化合物的各种代谢途径。这些 研究也对理解基本的 酶过程，烯醇和二烯醇化学，和进化 酶的专一性和分解代谢途径。耦合的遗传分析 在此提出的酶学研究为理解 当晶体结构变得可用时，其机制并探索其作用 在催化中的特定活性部位残基和特异性通过 未来的定点突变。除了解决这些问题之外 智力问题，这些研究最终可能会有应用 朝着解决当前有毒废物问题的方向前进。

Several microorganisms are able to use an aromatic hydrocarbon as a sole source of carbon and energy. This capability results from highly specialized pathways that convert the aromatic hydrocarbon to intermediates in the Krebs cycle. Initially, the aromatic hydrocarbon is converted to catechol or a substituted catechol. Subsequently, the catecholic compound is processed to the Krebs cycle by one of the many so- called meta-fission pathways. The battery of enzymes comprising these meta-fission pathways are rich in mechanistic, structural, and evolutionary questions and present an attractive strategy for the degradation of aromatic hydrocarbons in the environment. The long term goal of this research is to apply techniques from protein chemistry, molecular biology, and x-ray crystallography to study thoroughly the mechanism, structure, and evolution of the enzymes in two meta-fission pathways - the catechol and homoprotocatechuate meta-fission pathways. During this funding period, we will continue our studies on 4- oxalocrotonate tautomerase (4-OT), 4-oxalocrotonate decarboxylase (4-OD), and vinylpyruvate hydratase (VPH). All are in the catechol meta-fission pathway. Our major specific aims, listed in the order of priority, during this funding period will be to: (l) Examine further the "anchoring" hypothesis for 4-OT using substrate analogs; (2) Characterize the behavior of 4-OT with a potential acetylenic mechanism-based inhibitor and a potential affinity label; (3) Examine the role of proline-1 in the mechanism of 4-OT by the chemical synthesis and characterization of mutants; (4) Continue NMR studies of 4-OT as a prelude to a solution structure; (5) Determine the 13C kinetic isotope effects on the non-enzymatic and the 4-OD-mediated decarboxylation of 2-oxo-3-hexenedioate and compare the results to those previously determined for the decarboxylation of oxalacetate; (6) Determine the steric course of proposed allylic isomerization and a hydration reaction catalyzed by VPH if the configuration of the intermediate can be established; (7) Assign the stereochemistry at C-5 of the product of the VPH reaction; and (8) Examine the behavior of VPH with the acetylenic analog of its substrate. The proposed studies will address key mechanistic and evolutionary questions raised by what appears to be a common strategy used by the various meta-fission pathways to degrade catecholic compounds. These studies also have implications for the understanding of basic enzymological processes, enol and dienol chemistry, and the evolution of enzyme specificity and catabolic pathways. The genetic analysis coupled with the enzymological studies proposed herein set the stage to understand the mechanism as crystal structures become available and to probe the role of specific active site residues in catalysis and specificity through future site-directed mutagenesis. In addition to addressing these intellectual questions, these studies may ultimately have applications toward a solution of the incumbent toxic waste problem.