Flavoenzymes in Pyrimidine Metabolism

嘧啶代谢中的黄素酶

• 批准号: 7743760
• 负责人: BRUCE A PALFEY
• 金额: $ 27.76万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 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/7743760
• 关键词: Address; Bacteria; Basic Science; Behavior; Binding; Catalysis; Cells; Chemicals; Chemistry; Collaborations; Color; Communities; Cycloserine; DNA; Data Analyses; Deuterium; Dihydroorotate Dehydrogenase Inhibitor; Dihydroorotate dehydrogenase; Drops; Drug Delivery Systems; Electron Transport; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Flavins; Flavoproteins; Funding; Genetic; Goals; Grant; Heart; Human; Investigation; Isotopes; Kansas; Kinetics; Lead; Ligands; Malignant neoplasm of lung; Measures; Modeling; Mutagenesis; Outcome; Paper; Pathway interactions; Pharmaceutical Preparations; Productivity; Proteins; Protozoa; Publications; Published Comment; Publishing; RNA; Reaction; Reporting; Riboflavin; Role; Small Interfering RNA; Sorting - Cell Movement; Source; Specificity; Spectrum Analysis; Speed; Structure; Substrate Specificity; Thermotoga maritima; Thymidylate Synthase; Transfer RNA; Translating; Universities; Uracil; Vitamins; Work; Writing; base; cancer cell; cell growth; chemical reaction; design; dihydrouracil; dimer; drug candidate; drug development; inhibitor/antagonist; innovation; interest; medical schools; mutant; pathogenic bacteria; practical application; professor; pyrimidine metabolism; rapid growth; reaction rate (chemical); research study; single molecule

DESCRIPTION (provided by applicant): 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.

描述（由申请人提供）：嘧啶代谢至关重要，因此在化学水平上了解嘧啶代谢非常重要，并使其成为药物开发的绝佳靶点。我们将研究二氢乳清酸脱氢酶 (DHOD)、生物合成途径中的黄素依赖性酶以及进化相关的二氢尿苷合酶 (DUS) 的反应机制，这些酶在 tRNA 成熟过程中减少特定尿嘧啶。我们的目标是通过从表征过渡态到揭示催化动态行为等方式阐明反应机制和底物或配体特异性的起源。这些研究的结果将有助于酶抑制剂的设计，从而可能开发成有用的候选药物。过渡态结构是酶催化的核心。之前我们发现 1A 类和 2 类 DHOD 中黄素还原的过渡态之间存在显着差异，表明需要更高水平的理解。将通过测量 13C 和 15N 动力学同位素效应来探讨三类 DHOD 中黄素还原的过渡态。停流实验将用于确定氘同位素对 1B 类 DHOD 还原的影响。对 1B 类 DHOD 的补充停流和单分子研究将使我们能够剖析控制黄素化学、分子内电子转移和动力学的因素。我们已经发现了两种与 1A 类 DHOD 特异性结合的抑制剂，这种 1A DHOD 存在于一些病原细菌和原生动物中。我们的动力学和结构研究表明需要合成和研究一种新分子。然而，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受到抑制的功能性 1A 类突变体，以及相反地，受到抑制的 2 类突变体。有趣的酶将从热力学、动力学和结构上进行详细研究。相关化学由 DUS 进行，黄素蛋白在结构上与 DHOD 相关，并减少成熟 tRNA 中的特定尿嘧啶部分。二氢尿嘧啶的功能仍不确定，但其广泛存在表明其具有重要作用，并且最近已被证明在肺癌中具有重要作用。我们将确定所选模型 DUS 的底物特异性，并通过化学和生物物理手段探讨蛋白质和 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，这些化学反应会产生或修改 DNA 或 RNA（携带遗传信息的分子）的组成部分。专门干扰感染性细菌中这些重要反应的化合物可以用作药物。为了设计此类化合物，我们将通过观察与维生素相关的颜色变化来非常详细地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、它们在反应过程中如何移动、它们如何加速产物的合成以及如何阻止这些反应。