Structure and Mechanisms of PLP Dependent Lyases

PLP 依赖性裂解酶的结构和机制

• 批准号: 6951899
• 负责人: ROBERT STEPHEN PHILLIPS
• 金额: $ 3.88万
• 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/6951899
• 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) 含有吡哆醛-5‘-磷酸(PLP)的酶在生物中普遍存在，在氨基酸和胺的代谢中执行必要的反应。这些酶催化广泛的反应，包括外消旋、转氨化、α-和β-脱羧基、逆转-羟醛裂解、β-和伽马-消除和取代。这些酶反应的化学反应是由PLP辅因子控制的，而且PLP本身已经被证明执行这些反应中的许多，尽管速度非常低。因此，正是蛋白质环境赋予了这些酶典型的反应特异性和巨大的速度加速。碳-碳裂解酶，酪氨酸苯酚裂解酶(EC 4.1.99.2，TPL)，催化L酪氨酸的水解性β消除生成苯酚和丙酮酸铵。蛋氨酸-γ-裂解酶(EC 4.4.1.11，MGL)催化L-蛋氨酸的伽马消除反应生成甲硫醇和α-酮丁酸铵。我们工作的主要目标是在分子水平上对主要的结构-活性关系进行比较分析，这些关系是β-消除(TPL)和伽马-消除(MGL)裂解酶的特征。最耐人寻味的问题似乎是反应中心从底物的α-碳转移到β-碳。为了实现这一目标，将进行多项实验。将确定第三方物流反应中中间体的三维结构。根据底物结构的不同，将使用稳态和停流技术以及多重同位素效应研究来彻底分析TPL和MGL的催化机理。正在研究的酶是病原体的潜在药物靶点。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。