Studies on the mechanism of Pyridine Nucleotide Dependent Enzymes

本文的目的是为了更好地了解吡啶核苷酸在脱氢酶催化的氧化还原反应中作为辅酶以及在NAD糖水解酶催化的水解和ADP-核糖基化反应中作为底物的机制。这些研究将包括吡啶核苷酸依赖酶的分离和纯化，这些酶的化学、物理和动力学性质的研究，以及涉及底物和产物正确取向的结合过程的研究。吡啶和核苷酸的结构类似物将被合成，化学表征，并应用于酶过程中重要的选择性相互作用的研究。其中一些衍生物将被设计为荧光探针，而另一些将被设计为位点标记试剂或自杀底物。这些研究将采用分光光度法、荧光法、滴定法、液相色谱法和一些一般的生化和化学技术。辅酶类似物作为许多生化过程研究的实验工具的总体重要性是有据可查的。新提出的一系列具有独特性质的辅酶类似物将为生物化学研究提供额外的试剂。除了建议使用这些化合物来阐明流感嗜血杆菌中涉及NAD代谢的酶机制外，这些类似物应该在许多必需的吡啶核苷酸依赖性酶的机制研究中找到更广泛的应用。

The objectives here are to gain a better understanding of the mechanisms by which pyridine nucleotides function as coenzymes in dehydrogenase-catalyzed oxidation-reduction reactions and as substrates in NAD glycohydrolase-catalyzed hydrolyses and ADP- ribosylation reactions. These studies will include the isolation and purification of pyridine nucleotide dependent enzymes, the investigation of chemical, physical and kinetic properties of these enzymes and studies of the binding processes involved in the proper orientation of substrates and products. Structural analogs of the pyridine and nucleotides will be synthesized, chemically characterized, and applied in studies of selective interactions important to enzymic processes. Some of these derivatives will be designed as fluorescent probes while others will be designed to function as site-labeling reagents or suicide substrates. These studies will employ spectrophotometry, fluorimetry, titrimentry, liquid chromatography, and a number of general biochemical and chemical techniques. The overall importance of coenzyme analogs as experimental tools for he study of many biochemical processes is well-documented. The proposed new series of coenzyme analogs having unique properties will provide additional reagents applicable to biochemical studies. In addition to the proposed use of these compounds to elucidate enzymic mechanisms involved in NAD metabolism in H. influenzae, these analogs should find an even broader application in mechanistic studies of the many essential pyridine nucleotide-dependent enzymes.