ENZYMES INVOLVED IN THE METABOLISM OF VITAMIN B6 IN BRAI

BRAI 中参与维生素 B6 代谢的酶

基本信息

批准号：
3274847
负责人：
JORGE E CHURCHICH
金额：
$ 7.65万
依托单位：
UNIVERSITY OF TENNESSEE KNOXVILLE
依托单位国家：
美国
项目类别：
财政年份：
1980
资助国家：
美国
起止时间：
1980-07-01 至 1989-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3274847
关键词：
adenosine diphosphate adenosine triphosphate affinity chromatography brain metabolism chemical binding enzyme structure enzyme substrate enzyme substrate complex flavin mononucleotide fluorescence spectrometry nutrition related tag oxidoreductase pyridoxal phosphate pyridoxine stereochemistry vitamin B6 vitamin metabolism

项目摘要

The purpose of this research project is to study the mechanisms and regulation of the enzymes pyridoxal kinase and pyridoxine-5-p oxidase involved in the metabolism of vitamin B6 in brain tissues. The stereochemical course of pyridoxal kinase catalysed phosphoryl transfer will be examined using ATP, possessing a chiral Alpha-phosphorus, as a substrate. After analysis of the configuration of the product of the reaction, i.e., pyridoxal-5-p, one should be able to establish whether or not the phosphoryl group has been transferred directly between the two bound substrates. The topography of the catalytic site of the kinase will be studied using pyridoxyl derivatives of ATP and ADP, which are able to recognize the binding sites of the kinase and behave as competitive inhibitors with respect to ATP and pyridoxal. The bifunctional inhibitors display fluorescence properties that can be exploited to determine their mode of binding to the catalytic domains of the kinase. The regulation of the catalytic activity of pyridoxine-5-p oxidase by the product pyridoxal-5-p will be examined in detail. Experiments are designed to demonstrate that pyridoxal-5-p binds to a critical lysyl residue positioned in the catalytic domain of the oxidase. Pyridoxal kinase and pyridoxine-5-p oxidase interact in solution forming "clusters" of large molecular weight. The dissociation constant of such "enzymatic clusters" has been estimated to be lower than luM. The stability and stiochiometry of binding of the two cytosolic enzymes will be examined by a combination of hydrodynamic techniques, analytical ultracentrifugation and time resolved emission anisotropy. Affinity chromatography techniques using antibodies immobilized on protein-a sepharose will be used to detect the effects of the effectors pyridoxine-5-p and pyridoxal-5-p on the stability of the "enzyme clusters."

该研究项目的目的是研究机制和调节吡啶还毒激酶和吡ido醇-5-P氧化酶的调节参与脑组织中维生素B6的代谢。这吡啶毒素激酶催化磷酸化转移的立体化学过程将使用ATP检查，具有手性α-磷作为A 基材。在分析了产品的配置之后反应，即吡啶还毒-5-p，应该能够确定是否或不是直接在两者之间转移的磷酸基团绑定的底物。激酶的催化位点的地形将使用 ATP和ADP的吡啶毒素衍生物能够识别激酶的结合位点，并作为竞争性抑制剂与尊重ATP和吡啶还原。双功能抑制剂显示荧光特性可以利用以确定其模式与激酶的催化结构域结合。调节吡ido醇-5-P氧化酶的催化活性将详细检查产品吡啶-5-P。设计实验为了证明吡idox-5-p与临界赖氨酸残基结合位于氧化酶的催化结构域中。吡ido醇激酶和吡ido醇-5-P氧化酶在形成溶液中相互作用大分子量的“簇”。这样的解离常数估计“酶簇”低于LUM。这两种胞质酶结合的稳定性和稳定计量法将是通过流体动力技术的结合，分析性检查超速离心和时间分辨的发射各向异性。亲和力使用固定在蛋白质上的抗体的色谱技术-A Sepharose将用于检测效应子的影响吡ido醇-5-P和吡ido醇5-P对“酶簇”的稳定性。