ELECTROPHILIC CATALYSIS--ENZYMES AND MODELS

亲电催化——酶和模型

• 批准号: 2750011
• 负责人: JOHN A GERLT
• 金额: $ 21.24万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-02 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2750011
• 关键词: X ray crystallography; acidity /alkalinity; active sites; chemical elimination; chemical kinetics; chemical stability; crystallization; enol; enzyme mechanism; enzyme model; enzyme structure; enzyme substrate; enzyme substrate complex; flavin mononucleotide; hydro lyase; hydrogen bond; ionization; ionization constant; isomerase; mutant; oxidation; phenylacetates; site directed mutagenesis; stereochemistry; stop flow technique

DESCRIPTION (adapted from the applicant's abstract): Many enzyme- catalyzed reactions involve abstraction of a weakly acidic proton from the carbon adjacent to a carbonyl or carboxylic acid group (alpha-proton of a carbon acid) by a weakly basic active site general basic catalyst. An electrophilic (general acidic) catalyst positioned adjacent to the carbonyl group of the substrate so that an enolic intermediate can be stabilized by a short, strong hydrogen bond to the acid catalyst allows a quantitative understanding of the rates and mechanisms of these reactions. Dr. Gerlt will investigate the importance of electrophilic catalysis in three enzyme-catalyzed reactions: 1) the cycloisomerization of cis,cis-muconate catalyzed by muconate lactonizing enzyme I (MLE I); 2) the beta-elimination of water from acid sugars catalyzed by galactonate dehydratase (GalDH) and glucarate dehydratase (GlucDH); and 3) the FMN-dependent oxidation of S-mandelate catalyzed by S-mandelate dehydrogenase (MDH). MLE I, GalDH, and GlucDH are homologous to the structurally and mechanistically characterized mandelate racemase (MR). The proposed studies will increase our understanding of the rates and mechanisms of proton abstraction from carbon acids, in general, and beta-elimination reactions, in particular. Although both MR and MDH catalyze the abstraction of the alpha proton of S-mandelate to generate enolic intermediates, the active site architectures of MR and MDH are significantly different, e. g., the reaction catalyzed by MR but not MDH is absolutely dependent upon Mg2+. The investigator has been able to detect the enolic intermediate in the reaction catalyzed by MDH (but has been unable to do so in the reaction catalyzed by MR), so the structural factors important in the formation of this intermediate can be studied in the active site of MDH. He will also investigate the importance of matched pKa's for the donors and acceptors in anionic short, strong hydrogen bonds so that the requirements for the stabilization of enolic intermediates in enzyme active sites can be better understood.

描述（改编自申请人的摘要）：许多酶 - 催化反应涉及从中弱酸性质子的抽象 与羧酸或羧酸基相邻的碳（Alpha-proton 由弱基本活性位点一般基本催化剂的碳酸）。 与位于邻近的亲电（一般酸性）催化剂 基板的羰基，以便可以是含有的中间体 用短而强的氢键稳定在酸催化剂上可以 对这些速率和机制的定量理解 反应。 Gerlt博士将研究亲电的重要性 三种酶催​​化反应中的催化：1） CIS的环化，顺式粘胶酸酯通过粘液酸酯乳酸化催化 酶I（mle i）; 2）从酸性糖中取水的β-灭绝 通过半乳糖酸酯脱氢酶（GALDH）和葡萄糖脱氢酶催化 （glucdh）; 3）S-MandeLate催化的FMN依赖性氧化 由S-甲酸脱氢酶（MDH）。 mle I，galdh和glucdh是 与结构和机械特征的同源 Mandelate Racemase（MR）。拟议的研究将增加我们的 了解质子抽象的速率和机制 通常，碳酸尤其是β-淘汰反应。 尽管MR和MDH都催化了Alpha Proton的抽象 S-MandeLate生成活性位点生成enolic中间体 MR和MDH的体系结构显着不同，e。 g。， MR但非MDH催化的反应绝对取决于Mg2+。 研究人员能够检测到该中间的中间体 反应由MDH催化（但在反应中无法做到 由MR）催化，因此结构因素在形成中很重要 可以在MDH的活跃部位研究此中级。他会的 还要研究匹配PKA对捐助者的重要性， 阴离子短而强的氢键中的受体，以便 在酶中稳定中间体的要求 可以更好地理解活跃的站点。