MECHANISMS OF CARBON-CARBON LYASES AND KYNURENINASE

碳-碳裂解酶和犬尿氨酸酶的机制

• 批准号: 2181502
• 负责人: ROBERT STEPHEN PHILLIPS
• 金额: $ 11.98万
• 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 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2181502
• 关键词: acid base balance; acidity /alkalinity; alanine; aminoacid metabolism; carbon carbon lyase; carboxylation; catalyst; chemical structure function; chemical synthesis; electrolyte balance; enzyme mechanism; molecular pathology; phenols; protein sequence; pyridoxal phosphate; racemization; thermodynamics; tryptophanase; tyrosine analog

Enzymes containing the cofactor, pyridoxal phosphate, are ubiquitous in biology, performing essential functions in the metabolism of amino acids and amines. These enzymes catalyze a wide variety of reactions, including transaminations, racemizations, alpha- and beta-decarboxylations, retro-Aldol cleavages, beta- and gamma-eliminations and substitutions. The goal of our research is to understand, at the molecular level, the mechanisms of three of these enzymes, tryptophan indole-lyase, tyrosine phenol-lyase, and kynureninase. These enzymes catalyze unusual elimination reactions with carbon leaving groups, and thus require chemical steps involving the leaving groups in order for the reactions to proceed. We will synthesize and evaluate novel mechanism-based inhibitors and suicide substrates for these enzymes. In addition, we will prepare and examine a series of aza-analogues of substrates for tryptophan indole-lyase and tyrosine phenol-lyase. We will demonstrate the reversibility of the reaction of kynureninase using aryl esters to catalyze Claisen-type condensations with L-alanine. We will perform both steady-state and stopped-flow kinetic studies with these enzymes. For kynureninase, we will evaluate pH dependencies and isotope effects on the steady-state kinetics to evaluate the role of general acid/base catalysis. We will also examine in detail the effects of monovalent cations on the steady-state and pre-steady-state kinetic parameters for tryptophan indole-lyase and tyrosine phenol-lyase. These data will provide important information about the details of the enzymatic reaction mechanisms. We will clone and sequence the gene coding for tyrosine phenol-lyase from Citrobacter freundii. Then we will be able to determine the homology of tyrosine phenol-lyase with tryptophan indole-lyase. This will allow us to determine the evolutionary relationships between the enzymes with respect to structure, mechanism and regulation. Also, we will prepare mutants of tryptophan indole-lyase by sitedirected mutagenesis. These studies will allow us to evaluate the structural and catalytic roles of specific amino acid residues.

含有辅因子磷酸吡哆醛的酶普遍存在于 生物学，在氨基酸代谢中发挥重要功能 和胺类。这些酶催化多种反应，包括 转氨作用、外消旋作用、α-和β-脱羧作用， 逆羟醛裂解、β-和γ-消除和取代。这 我们研究的目标是在分子水平上了解 其中三种酶的机制：色氨酸吲哚裂解酶、酪氨酸 酚裂解酶和犬尿氨酸酶。这些酶催化异常消除 与碳离去基团反应，因此需要化学步骤 涉及离去基团以便反应进行。 我们将合成和评估基于机制的新型抑制剂 这些酶的自杀底物。此外，我们将准备并 检查一系列色氨酸吲哚裂解酶底物的氮杂类似物 和酪氨酸酚裂解酶。我们将证明其可逆性 犬尿氨酸酶使用芳基酯催化 Claisen 型反应 与L-丙氨酸缩合。 我们将进行稳态和停流动力学研究 这些酶。对于犬尿氨酸酶，我们将评估 pH 依赖性并 同位素对稳态动力学的影响来评估 一般酸/碱催化。我们还将详细研究其影响 单价阳离子对稳态和前稳态动力学的影响 色氨酸吲哚裂解酶和酪氨酸苯酚裂解酶的参数。这些 数据将提供有关酶详细信息的重要信息 反应机制。 我们将克隆并测序编码酪氨酸酚裂解酶的基因 弗氏柠檬酸杆菌。然后我们就可以确定同源性 酪氨酸苯酚裂解酶和色氨酸吲哚裂解酶。这将使我们能够 确定酶之间的进化关系 结构、机制和监管。此外，我们将准备突变体 通过定点诱变实现色氨酸吲哚裂解酶。这些研究将 使我们能够评估特定氨基的结构和催化作用 酸残留物。