REACTION SPECIFICITY OF PYRIDOXAL PHOSPHATE ENZYMES

磷酸吡哆醛酶的反应特异性

• 批准号: 6386627
• 负责人: Michael Toney
• 金额: $ 19.29万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-05-01 至 2003-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6386627
• 关键词: X ray spectrometry; alkali metals; apoenzymes; carboxylation; catalyst; chemical cleavage; decarboxylases; enzyme activity; enzyme mechanism; enzyme structure; transaminases; transamination

DESCRIPTION: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, and a fundamental, unresolved question that bears on the design of enzyme inhibitors 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, thereby providing a vehicle for incisive studies on the control of enzymatic reaction specificity. High resolution X-ray structures for DGD are available, and reveal two alkali metal ion-specific binding sites. One, near the active site, is responsible for the activating effects of large (K+, Rb+) and inhibitory effects of small (Na+, Li+) ions. The elucidation of the mechanisms by which DGD discriminates between and is catalytically controlled by alkali metals has very broad physiological significance. DGD is structurally representative of a medically important class of aminotransferases capable of acting alternately on primary amines and alpha-amino acids. The specific aims of this project are to test the hypotheses that: 1) C-C (decarboxylation) and C-H (transamination) bond cleavage occur via a single substrate binding subsite in the DGD active site, which activates these bonds by large stereoelectronic effects; 2) DGD specifically catalyzes oxidative vs. non-oxidative decarboxylation by proceeding through a concerted transition state in which CO2 loss and proton transfer occur simultaneously; 3) Active site structural changes in Ser80 and Tyr301 caused by exchange of activating for inhibitory ions control catalytic activity; 4) The ability of several aminotransferases to act in alternative half-reactions on primary amines and alpha-amino acids is largely determined by two active site residues, Glu215 and Va1244.

描述：磷酸吡哆醛 (PLP) 依赖性酶普遍存在于 氮代谢并催化许多医学上重要的转化。 作为一个群体，它们催化了极其广泛的反应，并且 与酶设计有关的一个基本的、未解决的问题 抑制剂是给定的脱辅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 快速催化脱羧和转氨作用的酶 正常的催化循环，从而为深入研究提供了工具 酶促反应特异性的控制。 高分辨率 X 射线 DGD 的结构可用，并揭示了两种碱金属离子特异性 结合位点。 其中一个位于活性位点附近，负责激活 大离子（K+、Rb+）的作用和小离子（Na+、Li+）的抑制作用。 DGD 区分 和 的机制的阐明 碱金属催化控制具有非常广泛的生理学 意义。 DGD 在结构上代表了具有重要医学意义的 能够交替作用于伯胺的一类转氨酶 和α-氨基酸。 该项目的具体目标是测试 假设：1）C-C（脱羧）和C-H（转氨基）键 裂解通过 DGD 活性中的单个底物结合亚位点发生 位点，通过大的立体电子效应激活这些键； 2）DGD 特异性催化氧化脱羧与非氧化脱羧 经历一个协调一致的过渡状态，其中二氧化碳损失和质子 转移同时发生； 3）Ser80活性位点结构变化 和 Tyr301 由激活离子控制与抑制离子控制交换引起 催化活性； 4) 几种转氨酶的作用能力 伯胺和α-氨基酸的替代半反应是 主要由两个活性位点残基 Glu215 和 Va1244 决定。