REACTION SPECIFICITY OF PYRIDOXAL PHOSPHATE ENZYMES

磷酸吡哆醛酶的反应特异性

• 批准号: 2910277
• 负责人: Michael Toney
• 金额: $ 2.09万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 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 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2910277
• 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：吡啶还毒素（PLP）依赖性酶无处不在 氮代谢并催化许多医学上重要的转化。 作为一个小组，他们催化了各种各样的反应，并且 一个基本的，未解决的问题，涉及酶的设计 抑制剂是给定的载酶如何决定独特的反应 特异性。 Dialkylglycine脱羧酶（DGD）是不寻常的PLP 酶在其中迅速催化脱羧基化和转移的酶 正常的催化循环，从而为 酶促反应特异性的控制。 高分辨率X射线 可用于DGD的结构，并揭示两个碱金属特异性 绑定位点。 一个，在活动地点附近，负责激活 大（K+，RB+）和小（Na+，Li+）离子的抑制作用的影响。 阐明DGD区分和IS的机制 由碱金属催化控制的生理非常广泛 意义。 DGD在结构上代表了医学上重要的 能够交替作用在原胺上的氨基转移酶类 和α-氨基酸。 该项目的具体目的是测试 假设：1）C-C（脱羧）和C-H（Transimination）键 通过DGD活性中的单个底物结合子线发生裂解 位点，通过大型立体电代效应激活这些键； 2）DGD 特异性地催化氧化与非氧化脱羧的催化 通过一致的过渡状态进行二氧化碳损失和质子的过渡状态 转移同时发生； 3）SER80中的主动位点结构变化 和Tyr301由激活抑制离子控制引起的 催化活性； 4）几种氨基转移酶起作用的能力 原代胺和α-氨基酸的替代半反应是 在很大程度上由两个活性位点残基GLU215和VA1244确定。