MECHANISMS OF PYRIDOXAL 5' PHOSPHATE DEPENDENT ENZYMES

吡哆醛 5 磷酸依赖性酶的机制

• 批准号: 2684914
• 负责人: ROBERT STEPHEN PHILLIPS
• 金额: $ 16.18万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-09-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2684914
• 关键词: X ray crystallography; alanine; aminoacid metabolism; carbon carbon hydrolase; carbon carbon lyase; carbon sulfur lyase; chemical synthesis; enzyme activity; enzyme inhibitors; enzyme mechanism; enzyme structure; kynurenine; molecular cloning; protein sequence; pyridoxal phosphate; site directed mutagenesis; transaminases; tryptophan; tyrosine

Enzymes containing pyridoxal-5'-phosphate (PLP), also known as vitamin B6, are ubiquitous in biology, performing essential reactions in metabolism of amino acids and amines. These enzymes catalyse a wide variety of reactions, including racemization, transamination, alpha- and beta- decarboxylation, retro-aldol cleavage, beta- and gamma-elimination and substitution. The central role of PLP-dependent enzymes in the metabolism of amino acids and amines, and the biosynthesis of important bioactive metabolites, makes them attractive targets for the design of mechanism- based inhibitors, which may be useful as drugs. The carbon-carbon lyases (tyrosine phenol-lyase and tryptophan indole-lyase) are bacterial enzymes which catalyse the hydrolytic beta-elimination of L-tyrosine or L- tryptophan to give, respectively, phenol or indole, and ammonium pyruvate. These enzymes are ideal models to study the relationship between structure and mechanisms in PLP enzymes. The structures and mechanisms of these enzymes will be studied by mutagenesis, X-ray crystallography, and pre- steady-state kinetics. Kynureninase catalyses the hydrolytic cleavage of L-kynurenine to give anthranilic acid and L-alanine. In eucaryotic organisms, the product of kynureninase, 3-hydroxyanthranilic acid, undergoes ring cleavage by a dioxygenase to give alpha-amino-beta- carboxymuconic semialdehyde, which can cyclize spontaneously to give quinolinic acid, a known neurotoxin. Inhibitors of kynureninase could thus be of value in the treatment of some serious neurological disorders, such as Huntington's chorea, ALS, stroke, epilepsy, and AIDS-related dementia. In order to aid in the design of new inhibitors, kynureninase will be cloned, sequenced, and the three dimensional structure determined. Last year, the product of nifS was found to be a PLP-dependent enzyme which catalyses the desulfurization of L-cysteine to give L-alanine and elemental sulfur. It has been proposed that this enzyme is required for the in vitro synthesis of iron-sulfur centers, which are necessary for electron transfer processes and are thus ubiquitous in biology. The mechanism of L-cysteine desulfurase will be studied by steady-state and pre-steady-state kinetic methods. The results of our continuing research are likely to make a significant contribution to the goal of improving health care for Americans in the future.

含有吡ido-5'-磷酸（PLP）的酶，也称为维生素B6， 在生物学上无处不在，在代谢中做出基本反应 氨基酸和胺。 这些酶催化了各种各样的 反应，包括种族化，跨动力，α-和β- 羧基化，复古 - 醛固醇裂解，β-和伽马脱离以及 替代。 PLP依赖性酶在代谢中的核心作用 氨基酸和胺，以及重要生物活性的生物合成 代谢物，使它们成为设计机制的吸引力 - 基于抑制剂，可能可作为药物有用。 碳碳裂解酶 （酪氨酸苯酚 - 洛酶和色氨酸吲哚酶）是细菌酶 这催化了L-酪氨酸或L-的水解β- 色氨酸分别给出苯酚或吲哚和丙酮酸铵。 这些酶是研究结构之间关系的理想模型 和PLP酶的机制。 这些结构和机制 酶将通过诱变，X射线晶体学和前 稳态动力学。 Kynureninase催化了水解裂解 l-京硝氨酸给出蒽和L-丙氨酸。 在桉树中 生物体，Kynureninase的产物，3-羟基雄性酸， 通过双加氧酶进行环裂，以给予α-氨基β- 羧基少量甲醛，它可以自发地循环以给予 喹啉酸，一种已知的神经毒素。 Kynureninase的抑制剂可以 因此，在治疗某些严重的神经系统疾病中具有价值 例如亨廷顿的唱片，ALS，中风，癫痫和与艾滋病有关的 失智。 为了帮助设计新抑制剂，kynureninase 将被克隆，测序和确定的三维结构。 去年，发现NIFS的乘积是PLP依赖性酶 这会催化L-半半胱氨酸的脱硫为L-丙氨酸和 元素硫。 已经提出，这种酶是必需的 铁硫心中心的体外合成是必要的 电子转移过程，因此在生物学中无处不在。 这 稳态和 稳态的态动力学方法。 我们继续研究的结果 可能为改进目标做出重大贡献 将来为美国人提供医疗保健。