MECHANISM OF PEP CARBOXYKINASE AND PYRUVATE CARBOXYLASE

PEP羧激酶和丙酮酸羧化酶的作用机制

• 批准号: 3290919
• 负责人: Vern L. Schramm
• 金额: $ 18.71万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-08-01 至 1992-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3290919
• 关键词: allosteric site; biotin; electron spin resonance spectroscopy; enzyme inhibitors; enzyme mechanism; enzyme structure; enzyme substrate; enzyme substrate analog; enzyme substrate complex; laboratory rat; lithium; manganese; mass spectrometry; nonradiation isotope effect; nuclear magnetic resonance spectroscopy; phosphoenolpyruvate carboxylase; pyruvate decarboxylase; vitamin deficiency

P-enolpyruvate carboxykinase and pyruvate carboxylase catalyze the formation of oxaloacetate from three-carbon substrates (P-enolpyruvate or pyruvate), nucleotide triphosphates (GTP or ATP) and carbon dioxide or bicarbonate. Both reactions require one metal ion in the formation of the nucleotide metal complex and one or more additional metal ions to form catalytically competent complexes. Both enzymes are essential steps in the pathway of gluconeogenesis from lactate or alanine in mammals. The steady-state kinetic properties of these enzymes have been characterized, however many of the important features of catalysis are poorly understood. One purpose of these studies will be to characterize the interactions of substrates and metals at the catalytic sites of the enzymes. The interactions of substrates or substrate analogues with metal ions will be measured in electron paramagnetic resonance experiments which can establish direct metal substrate contacts and quantitate metal water and metal enzyme contacts. The rates of formation and dissociation of enzyme-substrate complexes will be established by substrate trapping experiments using labeled substrates. Reversibility of catalytic steps will be estimated by positional isotopic exchange experiments which can be quantitated by nuclear magnetic resonance or mass spectrometry. Heavy-atom kinetic isotope effects will be used to investigate the effects of allosteric activation on pyruvate carboxylase. These experiments will attempt to establish coordination of the nucleotide-bound metal and the enzyme-bound metals in the catalytic sites of both enzymes. Phosphobiotin or carboxyphosphate will be implicated as the intermediate in the pyruvate carboxylase reaction. The rates of catalytic steps and their reversibility will allow analysis of commitments to catalysis. These can then be used to establish the mechanism by which the allosteric activator, acetyl-CoA, activates pyruvate carboxylase and the metal activator, Mn(II), activates P-enolpyruvate carboxykinase. Heavy-atom kinetic isotope effects may be able to distinguish between changes in the transition state structure and changes in rate-limiting step(s) of pyruvate carboxylase in response to the allosteric activator.

β-烯醇式丙酮酸羧酸激酶和丙酮酸羧基酶催化 从三碳底物(P-烯醇式丙酮酸或丙酮酸)生成草酰乙酸酯 丙酮酸)、核苷酸三磷酸(GTP或ATP)和二氧化碳或 小苏打。这两个反应都需要一种金属离子才能形成 核苷酸金属络合物与一个或多个附加金属离子形成 具有催化活性的络合物。这两种酶都是 哺乳动物中乳酸或丙氨酸的糖异生途径。这个 对这些酶的稳态动力学特性进行了表征， 然而，人们对催化的许多重要特征知之甚少。 这些研究的目的之一将是描述 酶催化部位的底物和金属。这个 底物或底物类似物与金属离子的相互作用 在电子顺磁共振实验中进行测量，可以确定 金属底物与金属水和金属酶的直接接触和定量 联系人。酶-底物的形成和解离速率 络合物将通过底物捕获实验建立，使用 标有标签的底物。催化步骤的可逆性将通过以下方式进行评估 位置同位素交换实验可以通过 核磁共振或质谱学。重原子动力学 同位素效应将被用来研究变构的影响 丙酮酸羧基酶的激活。 这些实验将试图建立协调的 核苷酸结合金属和催化部位的酶结合金属 两种酶的结合。磷酸核黄素或羧基磷酸盐将被牵连为 丙酮酸羧化反应中的中间体。这一比率 催化步骤及其可逆性将有助于对承诺进行分析。 为了催化。然后，这些可以用来建立机制，通过该机制 变构激活剂，乙酰辅酶A，激活丙酮酸羧基酶和 金属激活剂Mn(II)能激活P-烯醇式丙酮酸羧酸激酶。 重原子动力学同位素效应或许能够区分 过渡态结构的变化和限速的变化 丙酮酸羧化酶对变构激活剂的反应步骤(S)。

项目成果

Inactivation of chicken mitochondrial phosphoenolpyruvate carboxykinase by o-phthalaldehyde.

邻苯二甲醛灭活鸡线粒体磷酸烯醇丙酮酸羧激酶。

• 发表时间: 1991
• 期刊: The Journal of biological chemistry
• 作者: Chen,CY;Emig,FA;Schramm,VL;Ash,DE
• 通讯作者: Ash,DE

Mammalian and avian liver phosphoenolpyruvate carboxykinase. Alternate substrates and inhibition by analogues of oxaloacetate.

哺乳动物和禽类肝脏磷酸烯醇丙酮酸羧激酶。

• 发表时间: 1990
• 期刊: The Journal of biological chemistry
• 作者: Ash,DE;Emig,FA;Chowdhury,SA;Satoh,Y;Schramm,VL
• 通讯作者: Schramm,VL

Chemical modifications of chicken liver pyruvate carboxylase: evidence for essential cysteine-lysine pairs and a reactive sulfhydryl group.

鸡肝丙酮酸羧化酶的化学修饰：基本半胱氨酸-赖氨酸对和反应性巯基的证据。

• DOI: 10.1006/abbi.1993.1275
• 发表时间: 1993
• 期刊: Archives of biochemistry and biophysics
• 影响因子: 3.9
• 作者: Werneburg,BG;Ash,DE
• 通讯作者: Ash,DE

VO2+(IV) complexes with pyruvate carboxylase: activation of oxaloacetate decarboxylation and EPR properties of enzyme-VO2+ complexes.

• DOI: 10.1021/bi971282r
• 发表时间: 1997-11
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: B. Werneburg;D. Ash