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: 3290917
• 负责人: Vern L. Schramm
• 金额: $ 17.8万
• 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 1991-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3290917
• 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-烯醇丙酮酸羧激酶和丙酮酸羧化酶催化 从三碳底物（β-烯醇丙酮酸或 丙酮酸盐）、核苷酸三磷酸（GTP或ATP）和二氧化碳或 碳酸氢盐 这两种反应都需要一个金属离子来形成 核苷酸金属络合物和一种或多种另外的金属离子以形成 催化活性络合物。 这两种酶都是 在哺乳动物中从乳酸或丙氨酸的代谢途径。 的 这些酶的稳态动力学性质已经被表征， 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是描述以下因素的相互作用： 底物和金属在酶的催化位点。 的 底物或底物类似物与金属离子的相互作用将 在电子顺磁共振实验中测量， 直接金属基质接触并定量金属水和金属酶 联系人. 酶-底物的形成和解离速率 复合物将通过底物捕获实验建立， 标记底物。 催化步骤的可逆性将通过 位置同位素交换实验，其可通过以下方法定量： 核磁共振或质谱法。 重原子动力学 同位素效应将被用来研究变构的影响， 对丙酮酸羧化酶的活化。 这些实验将试图建立协调的 催化位点中的核苷酸结合金属和酶结合金属 这两种酶。 磷碘素或羧基磷酸盐将被牵连， 丙酮酸羧化酶反应的中间产物。 率 催化性步骤及其可逆性将有助于分析承诺 到催化。 然后，这些可以用来建立一种机制， 变构激活剂乙酰辅酶A激活丙酮酸羧化酶， 金属激活剂Mn（II）激活β-烯醇丙酮酸羧激酶。 重原子动力学同位素效应可能能够区分 过渡态结构的变化和速率限制的变化 丙酮酸羧化酶响应变构激活剂的步骤。