Reaction Specificity of Pyridoxal Phosphate Enzymes

磷酸吡哆醛酶的反应特异性

基本信息

批准号：
7582491
负责人：
Michael Toney
金额：
$ 30.5万
依托单位：
UNIVERSITY OF CALIFORNIA AT DAVIS
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-05-01 至 2012-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7582491
关键词：
4-Aminobutyrate aminotransferase Active Sites Address Alanine Alanine Racemase Amines Amino Acids Anabolism Apoenzymes Aspartate Binding Carboxy-Lyases Cell Wall Class Coenzymes Comparative Study Complex Decarboxylation Dependence Diaminopimelate decarboxylase Drug Delivery Systems Enzymes Equilibrium Free Energy Grant Isotopes Kinetics Knowledge Measures Metabolic Metabolism Modeling Mutagenesis Mutation Nitrogen Numbers Ornithine Decarboxylase Pharmaceutical Preparations Pharmacologic Substance Public Health Pyridoxal Phosphate Pyridoxamine Pyruvate Pyruvates Racemases Reaction Research Solvents Source Specificity Statistical Methods Temperature Testing Vancomycin Resistance Vitamin B6 aspartate 4-decarboxylase design inhibitor/antagonist mutant nitrogen metabolism phenylalanine (histidine) aminotransferase protonation research study serine racemase small molecule transamination

项目摘要

DESCRIPTION (provided by applicant): Pyridoxal phosphate (PLP) dependent enzymes are ubiquitous in nitrogen metabolism and catalyze many medically important transformations. As a group, they catalyze an extraordinarily wide variety of reactions. A fundamental question directly bearing on inhibitor design is how a given apoenzyme determines a unique reaction specificity. Dialkylglycine decarboxylase (DGD) is an unusual PLP dependent enzyme that rapidly catalyzes both decarboxylation and transamination in its normal catalytic cycle. This allows a detailed exploration of stereoelectronic effects, which are a primary mechanism for determining PLP reaction specificity. We will now analyze mechanistically critical active site residues of DGD. Alanine racemase (AlaR) is the prototypical PLP dependent racemase, which provides D-alanine for bacterial cell wall biosynthesis. Free energy profile determination from global analysis of progress curves will be extended with AlaR to include the temperature dependence of a mesophilic and thermophilic AlaR, and statistical methods will be developed that will allow model testing using global analysis. Free energy profiles will also be determined for several active site mutants. The determination of isotopic free energy profiles will be extended, providing the effects of deuteration on all elementary steps. Comparative studies on the reaction specificity of diaminopimelate decarboxylase and ornithine decarboxylase initiated during the last granting period will be expanded to determine the origins of reaction specificity differences between these homologous enzymes. A new project on aspartate beta-decarboxylase will be initiated to understand how the reaction specificity is controlled in the complex reaction sequence employed by this enzyme. Lastly, the electrophilic requirements of PLP enzymes will be determined with 15N NMR experiments in which the protonation state of active site nitrogens of PLP enzymes will be determined, by using coenzyme analogs with pyridoxamine pyruvate aminotransferase, by determinining EIEs on external aldimine formation and by measuring C-H pKa's of enzyme-bound substrates. PUBLIC HEALTH RELEVANCE: Medically important enzymes that utilize vitamin B6 to make metabolic reactions go faster will be studied in mechanistic detail to understand how this large class of enzymes controls which product is made from which substrate (i.e. how these enzymes control reaction specificity). Several vitamin B6 dependent enzymes are targets of currently employed pharmaceuticals, and several of the enzymes we will study are excellent drug targets. Our studies will provide the fundamental knowledge required to target these enzymes highly specifically with small molecule drugs.

描述（由申请人提供）：吡啶毒素磷酸盐（PLP）依赖性酶在氮代谢中无处不在，并催化许多医学上重要的转化。作为一个小组，他们催化了多种反应。直接涉及抑制剂设计的一个基本问题是给定的载酶如何决定独特的反应特异性。二烷基甘氨酸脱羧酶（DGD）是一种不寻常的PLP依赖性酶，在其正常催化循环中迅速催化脱羧和跨动力。这允许对立体电动效应进行详细探索，这是确定PLP反应特异性的主要机制。现在，我们将分析DGD的机械临界活性位点残基。丙氨酸种族酶（ALAR）是原型PLP依赖性种族酶，为细菌细胞壁生物合成提供D-丙氨酸。从全球进度曲线分析中确定的自由能曲线将与Alar扩展，以包括中嗜和嗜热的Alar的温度依赖性，并将开发统计方法，该方法将允许使用全局分析进行模型测试。还将确定几个活性位点突变体的自由能曲线。同位素自由能谱的确定将扩展，从而提供申静以及所有基本步骤的影响。关于在最后一个授予期间开始的二氨基二二酰脱羧酶和鸟嘌呤脱羧酶的反应特异性的比较研究将扩大，以确定这些同源酶之间反应特异性差异的起源。将启动关于天冬氨酸β-二羧化酶的新项目，以了解该酶采用的复杂反应序列中如何控制反应特异性。 Lastly, the electrophilic requirements of PLP enzymes will be determined with 15N NMR experiments in which the protonation state of active site nitrogens of PLP enzymes will be determined, by using coenzyme analogs with pyridoxamine pyruvate aminotransferase, by determinining EIEs on external aldimine formation and by measuring C-H pKa's of enzyme-bound substrates.公共卫生相关性：使用维生素B6使代谢反应变得更快的医学上重要的酶将进行机械详细的研究，以了解这种大类的酶控制着如何制成哪种产物的底物（即这些酶如何控制反应反应特异性）。几种维生素B6依赖性酶是当前使用的药物的靶标，我们将研究的几种酶是极好的药物靶标。我们的研究将提供针对这些酶的基本知识，以高度针对小分子药物。