Structure and Mechanisms of PLP Dependent Lyases

PLP 依赖性裂解酶的结构和机制

• 批准号: 6786504
• 负责人: ROBERT STEPHEN PHILLIPS
• 金额: $ 3.72万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6786504
• 关键词: Commonwealth of Independent States; X ray crystallography; active sites; aspartate; chemical kinetics; chemical reaction; decarboxylases; enzyme activity; enzyme mechanism; international cooperation; lyase; methionine; phenols; pyridoxal phosphate; site directed mutagenesis; structural biology; tyrosine

DESCRIPTION (provided by applicant) Enzymes containing pyridoxal-5'-phosphate (PLP) are ubiquitous in biology, performing essential reactions in metabolism of amino acids and amines. These enzymes catalyze a wide variety of reactions, including racemization, transamination, alpha- and beta-decarboxylation, retro-aldol cleavage, beta- and gamma-elimination and substitution. The chemistry of these enzymatic reactions is controlled by the PLP cofactor, and PLP alone has been shown to perform many of these reactions, albeit at very low rates. Therefore, it is the protein environment that confers the reaction specificity and enormous rate accelerations typical of these enzymes. The carbon-carbon lyase, tyrosine phenol-lyase (EC 4.1.99.2, TPL), catalyzes the hydrolytic beta-elimination of L-tyrosine to give phenol and ammonium pyruvate. Methionine-gamma-lyase (EC 4.4.1.11, MGL) catalyzes gamma-elimination reactions of L-methionine to give methylmercaptan and ammonium alpha-ketobutyrate. The main goal of our work is comparative analysis on the molecular level of the principal structure-activity relationships which are characteristic for beta-eliminating (TPL) and gamma-eliminating (MGL) lyases. The most intriguing problem seems to be the transfer of the reaction center from the alpha- to beta-carbon of the substrate. To achieve this goal, a number of experiments will be carried out. Three dimensional structures of intermediates in the reactions of TPL will be determined. Thorough analysis of the catalytic mechanisms of TPL and MGL, depending on the substrate structures, will be performed, using steady state and stopped-flow techniques and multiple isotope effect studies. The enzymes under study are potential drug targets in pathogens. In addition, both TPL and MGL may be useful in cancer therapy. Thus, knowledge of the structure and mechanism of these enzymes may lead to new pharmaceuticals.

描述（由申请人提供） 含有吡ido-5'-磷酸（PLP）的酶在生物学上无处不在，在氨基酸和胺代谢中进行基本反应。这些酶催化了各种反应，包括种族化，跨氨基和β-二羧化，复古 - 醛固醇裂解，β-和伽马脱落和替代。这些酶促反应的化学因素由PLP辅因子控制，仅PLP被证明可以执行许多此类反应，尽管速率非常低。因此，正是蛋白质环境赋予这些酶典型的反应特异性和巨大速率加速度。碳碳裂解酶，酪氨酸酚 - l- 4.1.99.2，TPL）催化L-酪氨酸的水解β-脱氧化，从而得到苯酚和丙酮酸铵。蛋氨酸 - γ-乙酰酶（EC 4.4.1.11，MGL）催化L-甲宁氨酸的γ-溶态反应，从而给出甲基 - 封装和α-酮丁酸铵。我们工作的主要目的是对主要结构活性关系的分子水平的比较分析，这些分子水平是β-β-溶解（TPL）和γ-渗透性（MGL）裂解酶的特征。最吸引人的问题似乎是反应中心从α-到底物的β-碳的转移。为了实现这一目标，将进行许多实验。将确定中间体在TPL反应中的三维结构。将对TPL和MGL的催化机理进行详尽的分析，具体取决于底物结构，使用稳态和停止流动技术以及多种同位素效应研究。所研究的酶是病原体的潜在药物靶标。此外，TPL和MGL都可能在癌症治疗中有用。因此，对这些酶的结构和机制的了解可能会导致新的药物。