Studies of the Mechanism of Pyridine Nucleotide Dependent Enzymes

9315654安德森本项目的目标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NADase、NMNase、核苷酸焦磷酸酶和腺苷二磷酸核糖基酶的底物的功能。将合成NAD、NADP、NMN和烟酰胺单核苷酸的结构类似物，进行化学表征，并应用于与吡啶核苷酸依赖的酶的选择性相互作用的研究中。将包括作为替代底物、可逆抑制剂、荧光探针和位点标记试剂的衍生物。该项目将包括对依赖于吡啶核苷酸的酶进行纯化，然后对其化学、物理和动力学性质进行表征，并鉴定参与催化、结合和调节过程的官能团。选择用于研究的酶包括NMN：ATP腺酰转移酶、NAD激酶和流感嗜血杆菌的部分脱氢酶，参与这种生物独特的NAD新陈代谢的酶，以及满足这种生物独特的NAD生长需要的酶，以及棕色固氮杆菌的葡萄糖6-磷酸脱氢酶，这是一种重要的酶，可以产生还原的吡啶核苷酸作为固氮的主要电子供体。这些研究将使用分光光度法、荧光法、滴定法、高效液相色谱法、电泳法、亲和层析法和一些常规的生化和化学技术。%通过涉及各种细胞营养物质的氧化反应，可以满足维持和支持活细胞生长的能量需求。这些氧化反应是酶催化的，在大多数情况下，涉及酶(催化蛋白质)，它们与两个常见的第二反应成分之一，即吡啶核苷酸辅酶一起发挥作用。这些辅酶是氧化过程所必需的有机化合物，因此对细胞新陈代谢至关重要。这个项目的目标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，几种催化这类反应的酶将通过各种分级技术进行纯化。作为纯蛋白质，这些酶将根据与吡啶核苷酸辅酶的特定相互作用进行研究。为了便于这些研究，辅酶的化学性质将通过制备天然辅酶的结构类似物来改变。结构类似物将包括旨在加速或抑制相关酶的衍生物。将注意特定的酶-辅酶相互作用，这些相互作用对于细胞内环境中的酶的代谢控制可能是重要的。酶将被选为研究对象，因为它们参与了重要的生物过程。一种这样的酶将从棕色固氮菌中获得，这是一种目前正在研究的微生物，因为它在固氮中起着重要作用。其他酶将从婴儿细菌性脑膜炎的病原体伊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 ***