Studies of the Mechanism of Pyridine Nucleotide Dependent Enzymes

9315654安德森该项目的目的是进一步了解吡啶核苷酸作为氧化还原反应的辅酶和作为NAD酶、NMN酶、核苷酸焦磷酸酶和ADP-核糖基化酶的底物的功能。NAD、NADP、NMN和烟酰胺单核苷酸的结构类似物将被合成，化学表征，并应用于与吡啶核苷酸依赖酶的选择性相互作用的研究。 包括设计为替代底物的衍生物、可逆抑制剂、荧光探针和位点标记试剂。 该项目将涉及吡啶核苷酸依赖性酶的纯化，然后对化学、物理和动力学特性进行表征，并确定参与催化、结合和调节过程的官能团。 选择用于研究的酶包括NMN：ATP腺苷酰转移酶、NAD激酶和流感嗜血杆菌的选定的脱氢酶，这些酶参与该生物体的独特NAD代谢，并且对于满足该生物体的独特NAD生长需求是必需的，以及棕色固氮菌的葡萄糖6-磷酸脱氢酶，一种对于产生还原吡啶核苷酸作为固氮的主要电子供体具有重要意义的酶。 这些研究将采用分光光度法、荧光法、滴定法、高效液相色谱法、电泳法、亲和色谱法和一些一般的生化和化学技术。 维持和支持植物生长的能量需求 活细胞可以通过涉及各种细胞营养物的氧化的反应来满足。 这些氧化反应是酶催化的，并且在大多数情况下，涉及与两种常见的第二反应组分之一（即吡啶核苷酸辅酶）结合起作用的酶（催化蛋白质）。 这些辅酶是氧化过程发生所必需的有机化合物，因此对细胞代谢至关重要。 该项目的目的是进一步了解这些辅酶参与氧化反应的方式。 在这些研究中，催化这种类型的反应的几种酶将通过各种分馏技术纯化。 作为纯蛋白质，这些酶将研究与吡啶核苷酸辅酶的特异性相互作用。 为了促进这些研究，将通过制备天然辅酶的结构类似物来改变辅酶的化学性质。 结构类似物将包括设计用于加速或抑制所涉及的酶的衍生物。 将注意力放在特定的酶-辅酶相互作用，这可能是重要的代谢控制的酶在其细胞内环境。 由于酶参与重要的生物过程，因此将选择酶进行研究。 一种这样的酶将从棕色固氮菌（Azotobacter vinelandii）获得，该微生物由于在固氮中的重要性而目前正在研究中。 其他酶将从婴儿细菌性脑膜炎的病原体流感嗜血杆菌中获得 ***

9315654 Anderson The objective of this project are to gain further understanding of the functioning of pyridine nucleotides as coenzymes for oxidation- reduction reactions and as substrates for NADases, NMNases, nucleotide pyrophosphatases, and ADP-ribosylases Structural analogs of NAD, NADP, NMN, and nicotinamide mononucleotide will be synthesized, chemically characterized, and applied in studies of selective interactions with pyridine nucleotide dependent enzymes. Included will be derivatives designed as alternate substrates, reversible inhibitors, fluorescent probes, and site-labeling reagents. The project will involve the purification of pyridine nucleotide dependent enzymes followed by characterization with respect to chemical, physical and kinetic properties, and identification of functional groups involved in catalysis, binding and regulatory processes. Enzymes chosen for study include the NMN:ATP adenylytransferase, NAD kinase, and selected dehydrogenases of Haemophilus influenzae, enzymes involved in the unique NAD metabolism of this organism, and essential for meeting the unique NAD growth requirement of this organism, and the glucose 6- phosphate dehydrogenase of Azotobacter vinelandii, an enzyme of importance for producing reduced pyridine nucleotides as primary electron donors for nitrogen fixation. These studies will employ spectrophotometry, fluorimetry, titrimetry, HPLC, electrophoresis, affinity chromatography, and a number of general biochemical and chemical techniques.%%% The energy requirement for the maintenance and support of growth of living cells can be met through reactions involving the oxidation of various cell nutrients. These oxidative reactions are enzyme- catalyzed and in most cases, involve enzymes (catalytic proteins) that function in conjunction with one of two common second reaction components, namely, the pyridine nucleotide coenzymes. These coenzymes are organic compounds that are essential for the oxidative proce sses to occur and therefore, are of prime importance for cell metabolism. the objective of this project is to gain further understanding of the way in which these coenzymes participate in the oxidative reactions. In these studies, several enzymes that catalyze this type of reaction will be purified through various fractionation techniques. As pure proteins, these enzymes will be studied with respect to specific interactions with the pyridine nucleotide coenzymes. To facilitate these studies, the chemical properties of the coenzymes will be altered by preparing structural analogs of the natural coenzymes. The structural analogs will include derivatives designed to accelerate or inhibit the enzymes involved. Attention will be paid to specific enzyme-coenzyme interactions that could be impotant for metabolic control of the enzymes in their intracellular environment. Enzymes will be chosen for study due to their involvement in important biological processes. One such enzyme will be obtained from Azotobacter vinelandii, a microorganism currently under study because of is importance in nitrogen fixation. Other enzymes will be obtained from Haemophilus iinfluenzae, the causative agent of bacterial meningitis in infants ***