Mechanistic enzymology of quinolinic acid biosynthesis

描述（由申请人提供）：烟酰胺腺嘌呤二核苷酸（NAD）及其磷酸盐（NADP）是所有生命系统中必需的氧化还原辅助因子。令人惊讶的是，这种辅助因子的关键催化部分，喹啉酸衍生的吡啶环的生物合成仍然知之甚少。我们最近发现了一种细菌系统，它含有将色氨酸转化为喹啉酸所需的酶。与真核酶形成鲜明对比的是，所有细菌酶作为可溶性稳定酶都在高水平上过表达，这为该系统的详细表征打开了大门。本文介绍了色氨酸双加氧酶、甲酰基犬尿氨酸甲酰胺酶、犬尿氨酸单加氧酶、犬尿氨酸酶和羟氰氨酸双加氧酶的机制研究。这些研究将包括结构研究、每种酶的预稳态动力学表征、设计用于捕获反应中间体的底物类似物的合成和测试以及ESR研究。色氨酸到喹啉酸途径的中间体在许多生物过程中起着重要作用。犬尿氨酸是犬尿酸的前体，是谷氨酸受体的拮抗剂。在艾滋病相关痴呆、亨廷顿氏病和肝性脑病患者中，喹啉酸和3-羟基犬尿氨酸浓度均升高。3-Hydroxykynurenine诱导纹状体神经元凋亡。这种中间体在眼睛里也起着过滤器的作用，保护视网膜免受紫外线的伤害。3-羟基犬尿氨酸介导的交联反应可能在白内障形成中起作用。对喹啉酸生物合成酶的机理研究将有助于确定控制这些疾病状态的小分子抑制剂。

DESCRIPTION (provided by applicant): Nicotinamide adenine dinucleotide (NAD) and its phosphate (NADP) are essential redox cofactors in all living systems. Surprisingly, the biosynthesis of the key catalytic part of this cofactor, the quinolinic acid derived pyridine ring, is still poorly understood. We have recently discovered a bacterial system containing the enzymes required for the conversion of tryptophan to quinolinate. In dramatic contrast to the eukaryotic enzymes, all of the bacterial enzymes overexpress at a high level as soluble stable enzymes, opening up this system for detailed characterization. In this proposal, we describe mechanistic studies on tryptophan dioxygenase, formylkynurenine formamidase, kynurenine monooxygenase, kynureninase and hydroxyanthranilate dioxygenase. These studies will include structural studies, the characterization of the presteady state kinetics of each enzyme, the synthesis and testing of substrate analogs designed to trap reaction intermediates and ESR studies. The tryptophan to quinolinic acid pathway intermediates play an important role in several biological processes. Kynurenine is a precursor to kynurenic acid, an antagonist of the glutamate receptor. Both quinolinic acid and 3-hydroxykynurenine concentrations are elevated in patients with AIDS related dementia, Huntington's disease and hepatic encephalopathy. 3-Hydroxykynurenine induces apoptosis of neurons prepared from rat striatum. This intermediate also functions as a filter in the eye, protecting the retina from the damaging effects of UV-light. Crosslinking reactions mediated by 3-hydroxykynurenine may play a role in cataract formation. Mechanistic studies on the quinolinic acid biosynthetic enzymes will facilitate the identification of small molecule inhibitors to control these disease states.