ENZYMES OF THE META-FISSION PATHWAY

本研究的长期目标是应用蛋白质化学、分子生物学和X射线晶体学技术，深入研究儿茶酚和高原儿茶酸酯两条亚裂变途径中酶的机制、结构和进化。这些研究对于理解基本酶机制、烯醇和二烯醇化学以及酶特异性和分解代谢途径的进化具有意义。除了解决这些知识问题外，这些研究还有助于生物补救工作。在此资助期间，我们将继续我们的研究4-巴豆酸互变异构酶（4-OD），4-巴豆酸脱羧酶（4- OD）和乙烯基丙酮酸水合酶（VPH），三个连续的酶中发现的儿茶酚易位途径。拟议的研究将解决关键的机制和进化问题所提出的似乎是一个共同的儿茶酚亚裂变途径。拟议的研究将解决关键的机制和进化途径提出了什么似乎是一个共同的战略所使用的各种元裂变途径降解儿茶酚化合物。我们的主要具体目标（按优先顺序列出）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）使用编码和非编码氨基酸描述4-OT中活性位点丝氨酸的作用;（3）描述控制4-OT中催化碱基反应性的其他因素;（4）确定13 C动力学同位素对2-氧代-3-己烯二酸的非酶促和酶促脱羧的影响，作为pH和介质的函数;（5）研究底物通道是否涉及4-ODP/VPH复合物;（6）合成和评价VPH的潜在抑制剂;和（7）进行4- OD/VPH和OHED水合酶的晶体学研究。这些研究将解决关于这些酶的长期问题以及在前一个资助期提出的新问题。在这个过程中，他们将解决有关儿茶酚代谢途径的酶的主要进化问题。这些研究的结果作为与其他Meta裂变途径的酶进行比较的基础，以及未来实验和最终特异性和活性的遗传操作的基础。

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.