Flavoenzymes in Pyrimidine Metabolism

描述（由申请人提供）：嘧啶代谢至关重要，因此在化学水平上理解它非常重要，并使其成为药物开发的极好靶点。我们将研究二氢乳清酸脱氢酶（DHOD）的反应机制，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脱氢酶（DUS），其在tRNA成熟过程中减少特定的尿嘧啶。我们的目标是阐明反应机制和底物或配体特异性的起源，从表征过渡态到揭示催化中的动力学行为。这些研究的结果将有助于酶抑制剂的设计，这可能会发展成为有用的候选药物。过渡态结构是酶催化的核心。以前，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，这表明需要更高水平的理解。通过测量13C和15N动力学同位素效应，将探测这三类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。互补的停流和单分子研究1B类DHOD将使我们能够剖析控制黄素，分子内电子转移和动力学的化学因素。我们已经发现了两种抑制剂，其特异性结合于在一些病原性细菌和原生动物中存在的1A类DHOD。我们的动力学和结构研究表明，一个新的分子进行合成和研究。然而，我们的抑制剂不与2类酶结合的原因仍然是一个谜。随机诱变将用于产生不再受抑制的功能性1A类突变体，相反，产生受抑制的2类突变体。感兴趣的酶将详细研究的化学，动力学和结构。相关化学由DUS进行，DUS是结构上与DHOD相关并减少成熟tRNA中特异性尿嘧啶部分的黄素蛋白。二氢尿嘧啶的功能仍不确定，但其广泛存在表明其重要作用，最近已显示其在肺癌中的重要性。我们将确定选定的模型DUSs的底物特异性，并通过化学和生物物理手段探测蛋白质和tRNA的相互作用。 维生素B2在某些蛋白质加速化学反应时转移电子，这些化学反应产生或修改了DNA或RNA的结构单元，这些分子携带遗传信息。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。为了设计这样的化合物，我们将通过观察与维生素相关的颜色变化，以非常高的细节水平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，以及这些反应如何被阻止。

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.