MECHANISMS OF CARBON-CARBON LYASES AND KYNURENINASE

含有辅因子磷酸吡哆醛的酶普遍存在于 生物学的，在氨基酸新陈代谢中发挥重要作用的 还有胺。这些酶催化各种各样的反应，包括 转氨基，外消旋，α-和β-脱羧基， 复古-羟醛裂解，β-和伽马-消除和取代。这个 我们研究的目标是在分子水平上了解 色氨酸吲哚裂解酶、酪氨酸三种酶的作用机制 苯酚裂解酶和犬尿氨酸酶。这些酶催化不寻常的消除 与碳离开基团的反应，因此需要化学步骤 让离开的群体参与进来，以使反应继续进行。 我们将合成和评估新的基于机理的抑制剂和 这些酶的自杀底物。此外，我们还将准备和 检测色氨酸吲哚裂解酶底物的一系列氮杂类似物 和酪氨酸苯酚裂解酶。我们将演示 犬尿氨酸酶与芳香酯的反应催化Claisen型 与L丙氨酸的缩合反应。 我们将同时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依赖性和 同位素效应对稳态动力学的评价作用 一般的酸/碱催化。我们还将详细研究 一价阳离子对稳态和稳态前动力学的影响 色氨酸吲哚裂解酶和酪氨酸苯酚裂解酶参数。这些 数据将提供有关酶的细节的重要信息 反应机理。 我们将克隆和测序编码酪氨酸苯酚裂解酶的基因。 弗劳迪柠檬酸杆菌。然后我们就能确定它们的同源性 酪氨酸苯酚裂解酶和色氨酸吲哚裂解酶。这将使我们能够 以尊重的态度确定酶之间的进化关系 要结构、机制、规范。此外，我们还将准备突变体 色氨酸吲哚裂解酶的定点突变。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。

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.