ENZYMES OF THE META-FISSION PATHWAY

这项研究的长期目标是应用蛋白质化学、分子生物学和X射线结晶学的技术，深入研究两种元分裂途径-儿茶酚和高原儿茶酸盐的元分裂途径-儿茶酚和高原儿茶酸的元分裂途径中的酶的机制、结构和进化。这些研究对于理解酶的基本机制、烯醇和二烯醇化学以及酶的特异性和分解代谢途径的进化具有重要意义。除了解决这些智力问题外，这些研究还可能有助于生物修复工作。在这段资助期间，我们会继续研究邻苯二酚代谢途径中发现的三种顺序酶--4-草酰基-4-草酰基-4-丙酮酸互变互换酶(4-OD)、4-草酰基-4-丙酮酸脱羧酶(4-OD)和乙烯基丙酮酸水合酶(VPH)。拟议的研究将解决儿茶酚代谢裂变途径中常见的关键机制和进化问题。拟议的研究将解决关键的机制和进化途径，这些途径似乎是各种代谢裂变途径用来降解儿茶酚化合物的共同策略。在这一资助期间，我们的主要目标是：(1)合成潜在的4-OT二羧化抑制剂作为晶体结构的配体；(2)利用编码和非编码氨基酸描述活性部位精氨酸在4-OT中的作用；(3)描述控制4-OT中催化碱活性的其他因素；(4)确定~(13)C动力学同位素对2-氧代-3-己二酸酯的非酶和酶脱羧反应的影响，作为pH和介质的函数；(5)研究底物通道是否参与4-ODPVPH复合体；(6)合成和评价潜在的VPH抑制剂；(7)进行4-OD/VPH和OHED水合酶的结晶学研究。这些研究将解决关于这些酶的长期存在的问题，以及在前一个资助期提出的新问题。在这个过程中，他们将解决与儿茶酚代谢途径的酶有关的主要进化问题。这些研究的结果作为与其他代谢途径的酶进行比较的基础，也为未来的实验以及最终的特异性和活性的遗传操作奠定了基础。

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-the catechol and homoprotocatechuate meta-fission pathways. Such studies have implications for the understanding for the understanding of basic enzyme mechanisms, enol and dienol chemistry , and the evolution of enzyme specificity and catabolic pathways. In addition to addressing these intellectual questions, these studies may assist bio-remediation efforts. During this funding period, we will continue our studies on 4- oxalocrotonate tautomerase (4-OD), 4-oxalocrotonate decarboxylase (4- OD) and vinylpyruvate hydratase (VPH), three sequential enzymes found in the catechol metafission pathway. The proposed studies will address key mechanistic and evolutionary question raised by what appears to be a common in the catechol meta-fission pathway. The proposed studies will address key mechanistic and evolutionary pathways raised by what appears to be a common strategy used by the various meta-fission pathways to degrade catecholic compounds. Our major specific aims, listed in the order of priority, during this funding period will be to: (1) synthesize potential dicarboxylated inhibitors of 4- OT as ligands for crystal structures; (2) delineate the roles of the active site arginines in 4-OT using coded and non-coded amino acids; (3) delineate additional factors governing the reactivity of the catalytic base in 4-OT; (4) determine the 13C kinetic isotope effects on the non- enzymatic and enzymatic decarboxylation of 2-oxo-3-hexenedionate as a function of pH and medium; (5) investigate whether substrate channeling is involved in the 4-ODP/VPH complex; (6) synthesize and evaluate potential inhibitors of VPH; and (7) pursue crystallographic studies of 4- OD/VPH and OHED hydratase. These studies will address long-standing questions about these enzymes as well as new questions raised in the previous funding period. In the process, they will address major evolutionary questions concerning the enzymes of the catechol metafission pathway. The results from these studies serve as a basis for comparison to t he enzyme of other meta- fission pathways as well as the foundation for future experiments and ultimately the genetic manipulations of specificity and activity.