Flavoenzymes in Pyrimidine Metabolism

嘧啶代谢中的黄素酶

基本信息

批准号：
8048347
负责人：
BRUCE A PALFEY
金额：
$ 5.71万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-01-01 至 2011-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8048347
关键词：
Address Animal Welfare Basic Science Behavior Bibliography Binding Catalysis Cells Chemicals Chemistry Collaborations Communities Country Cycloserine Data Analyses Deuterium Dihydroorotate Dehydrogenase Inhibitor Dihydroorotate dehydrogenase Drops Drug Delivery Systems Electron Transport Environment Environmental Impact Enzyme Inhibitor Drugs Enzyme Inhibitors Enzymes Equipment Flavins Flavoproteins Funding Goals Grant Heart Human IACUC International Investigation Isotopes Kansas Kinetics Lead Ligands Malignant neoplasm of lung Measures Modeling Mutagenesis Outcome Paper Pathway interactions Principal Investigator Productivity Proteins Protozoa Publications Published Comment Publishing Reaction Reporting Research Research Ethics Committees Resources Role Small Interfering RNA Sorting - Cell Movement Source Specificity Spectrum Analysis Structure Substrate Specificity Thermotoga maritima Thymidylate Synthase Transfer RNA Translating Universities Uracil Vertebrates Work Writing abstracting base cancer cell cell growth design dihydrouracil dimer drug candidate drug development expiration human subject inhibitor/antagonist innovation interest medical schools mutant pathogenic bacteria practical application professor programs pyrimidine metabolism rapid growth research study single molecule

项目摘要

Pyrimidine metabolism is vital, making it important to understand at the chemical level and making it an excellent target for drug development. We will investigate the reaction mechanisms of dihydroorotate dehydrogenases (DHODs), flavin-dependent enzymes in the biosynthetic pathway, and the evolutionarily related dihydrouridine synthases (DUSs), which reduce specific uracils during the maturation of tRNA. Our goal is to elucidate reaction mechanisms and origins of substrate or ligand specificity in ways ranging from characterizing transition states to uncovering dynamic behavior in catalysis. The results of these studies will facilitate the design of enzyme inhibitors, which may be developed into useful drug candidates. Transition state structures are at the heart of enzymatic catalysis. Previously we found significant differences between the transition states for flavin reduction in Class 1A and Class 2 DHODs, indicating the need for a higher level of understanding. The transition states for flavin reduction in the three classes of DHODs will be probed by measuring 13C and 15N kinetic isotope effects. Stopped-flow experiments will be used to determine deuterium isotope effects on the reduction of a Class 1B DHOD. Complementary stopped-flow and single-molecule studies on a Class 1B DHOD will enable us to dissect factors controlling the chemistry at the flavins, intramolecular electron transfer, and dynamics. We have already discovered two inhibitors that bind specifically to Class 1A DHODs which occurs in some pathogenic bacteria and protozoa. Our kinetic and structural studies suggest a new molecule to be synthesized and studied. However, the reason that our inhibitors do not bind to Class 2 enzymes remains an enigma. Random mutagenesis will be used to create functional Class 1A mutants that are no longer inhibited, and conversely, Class 2 mutants that are inhibited. Interesting enzymes will be studied in detail thermodynamically, kinetically, and structurally. Related chemistry is performed by the DUSs, flavoproteins which are structurally related to DHODs and reduce specific uracil moieties in maturing tRNA. The function of dihydrouracil remains uncertain, but its widespread occurrence suggests an important role, and it has recently been shown to be important in lung cancer. We will determine the substrate specificities of selected model DUSs and probe the interactions of the protein and tRNA by chemical and biophysical means. Vitamin B2 transfers electrons when certain proteins speed chemical reactions that create or modify the building-blocks of DNA or RNA the molecules which carry genetic information. Compounds that specifically interfere with these vital reactions in infectious bacteria could be used as drugs. In order to design such compounds, we will study the reactions of several proteins at a very high level of detail by observing the color changes associated with the vitamin. Our studies of the rates of the chemical reactions will identify important parts of the proteins, how they move during reactions, how they speed the synthesis of products, and how these reactions might be blocked.

嘧啶的代谢至关重要，因此在化学水平上了解并成为药物开发的绝佳目标非常重要。我们将研究生物合成途径中的二氢肽脱氢酶（DHOD），黄素依赖性酶以及进化相关的二氢丙啶合酶（DUSS）的反应机制，这些酶在TRNA成熟过程中降低了特定的尿素。我们的目标是以从表征过渡状态到催化中的动态行为的方式来阐明底物或配体特异性的反应机制和起源。这些研究的结果将促进酶抑制剂的设计，这些酶抑制剂可能会发展为有用的候选药物。过渡状态结构是酶促催化的核心。以前，我们发现过渡态在1A类和2类DHOD中减少黄素的过渡状态之间存在显着差异，这表明需要更高的理解水平。通过测量13C和15N动力学同位素效应，将探测三类DHOD的黄素减少的过渡状态。停止流量实验将用于确定同位素对1B类DHOD的还原的影响。对1B类DHOD的互补停止流量和单分子研究将使我们能够解剖控制黄素的化学因素，分子内电子转移和动力学。我们已经发现了两个在某些致病细菌和原生动物中特异性结合的抑制剂。我们的动力学研究和结构研究提出了合成和研究的新分子。但是，我们的抑制剂不与2类酶结合的原因仍然是一个谜。随机诱变将用于创建不再抑制的功能性1a突变体，而相反，是被抑制的2类突变体。有趣的酶将在热力学，动力学和结构上进行详细研究。相关的化学是由杜斯（Duss）进行的，即在结构上与DHOD相关的黄素蛋白，并减少成熟tRNA中的特定尿嘧啶部分。二氢酸的功能仍然不确定，但其广泛的发生表明了重要作用，并且最近已证明它在肺癌中很重要。我们将通过化学和生物物理手段来确定选定模型duss的底物特异性，并探测蛋白质和tRNA的相互作用。当某些蛋白质加速化学反应时，维生素B2会转移电子，从而创建或修改DNA或RNA的建筑块，携带遗传信息的分子。特异性干扰感染细菌中这些重要反应的化合物可以用作药物。为了设计这种化合物，我们将通过观察与维生素相关的颜色变化来研究几种蛋白质的反应。我们对化学反应速率的研究将确定蛋白质的重要部分，它们在反应过程中的移动，如何加快产品的合成以及如何阻止这些反应。