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.

含有辅助因子吡啶多毒素的酶无处不在 生物学，在氨基酸的代谢中执行基本功能 和胺。这些酶催化了多种反应，包括 跨性别，种族化，α-和β-羧基化， 复古 - 醛固醇的切割，β-和γ-释放和取代。这 我们研究的目标是在分子水平上了解 这些酶的三种机制，色氨酸吲哚 - 酪氨酸 苯酚 - 乙醇酶和kynureninase。这些酶催化了异常消除 与碳离开组的反应，因此需要化学步骤 涉及离开小组以进行反应。 我们将合成并评估新型基于机制的抑制剂，并 这些酶的自杀底物。此外，我们将准备并 检查色氨酸吲哚 - 赖斯赖斯的一系列AZA-ANALOGUE 和酪氨酸酚酶。我们将证明 Kynureninase的反应使用芳基酯催化Claisen型 L-丙氨酸的凝结。 我们将通过 这些酶。对于Kynureninase，我们将评估pH依赖性和 同位素对稳态动力学的影响，以评估 一般酸/碱催化。我们还将详细研究 在稳态和稳态的动力学上的单价阳离子 色氨酸吲哚 - 乙醇酶和酪氨酸酚酶的参数。这些 数据将提供有关酶促细节的重要信息 反应机制。 我们将克隆并对编码酪氨酸酚 - lyase的基因进行测序 柠檬酸菌Freundii。然后，我们将能够确定 带有色氨酸吲哚酶的酪氨酸酚酶。这将使我们能够 确定酶之间的进化关系 结构，机制和调节。另外，我们将准备 色氨酸吲哚 - 通过置换诱变。这些研究会 允许我们评估特定氨基的结构和催化作用 酸残基。