KINETIC INVESTIGATION OF PYRUVATE CARBOXYLASE

丙酮酸羧化酶的动力学研究

• 批准号: 8168938
• 负责人: WILLIAM Wallace CLELAND
• 金额: --
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8168938
• 关键词: Acetyl Coenzyme A; Affect; Aspartate; Bicarbonates; Binding; Biological Assay; Biotin; Biotin carboxylase; Buffers; Carbon Dioxide; Carboxyltransferases; Computer Retrieval of Information on Scientific Projects Database; Enzymes; Firefly Luciferases; Funding; Grant; Holoenzymes; Institution; Investigation; Kinetics; Label; Length; Ligase; Location; Metabolic; MgATP; Movement; N1' -carboxybiotin; Oxaloacetates; Pyruvate; Pyruvate Carboxylase; Pyruvates; Reaction; Research; Research Personnel; Resources; Roentgen Rays; Role; Solutions; Source; Structure; System; United States National Institutes of Health; analog; carbonic acid, monoanhydride with phosphoric acid, ion(2-); carboxylate; formamide; inhibitor/antagonist; inorganic phosphate; mutant; tetrabutylammonium

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The purpose of this project is to use the structure of the full length biotin-containing pyruvate carboxylase holoenzyme, which we have recently solved, to determine the details of the catalytic mechanism of this important metabolic enzyme. Our specific aims are: 1) Use kinetic studies of wild type and key mutant enzymes to study the mechanism of the biotin carboxylase domain where MgATP and bicarbonate carboxylate biotin. Further X-ray structures will be obtained of mutants and with bound reactants other than MgATP. 2) Use kinetic studies of wild type and key mutant enzymes to study the mechanism of the carboxyl transferase domain where carboxybiotin converts pyruvate to oxaloacetate. Further X-ray structures will be obtained of mutants and with bound analogs of pyruvate.3) To clarify the role of acetyl-CoA as an allosteric activator, X-ray crystal structures will be determined in its absence as well as its presence. Structures will be determined with enzymes from sources where acetyl-CoA is not an activator. Structures will also be determined in the presence of aspartate, an allosteric inhibitor, to determine where it binds and how it affects the structural changes caused by acetyl-CoA.4) Investigation of the interdomain movement of biotin and carboxybiotin will be carried out using 1D and 2D NMR. [1-15N]-biotin and the methyl and acetyl analogs will be covalently attached to the enzyme using biotin ligase and a biotin auxotroph system. This label will allow us to probe the location and to what extent biotin is present in each domain.5) Carboxyphosphate will be synthesized by saturating a solution of tris[tetrabutylammonium] phosphate in dimethyl formamide with CO2. A small amount of this solution will be mixed in a stopped flow apparatus with enzyme, Mg2+, ADP and buffer and the resulting mixture analyzed for ATP formation with a firefly luciferase assay. If ATP is formed, this will establish carboxyphosphate as an intermediate in the reaction.The purpose of this project is to use the structure of the full length biotin-containing pyruvate carboxylase holoenzyme, which we have recently solved, to determine the details of the catalytic mechanism of this important metabolic enzyme. Our specific aims are: 1) Use kinetic studies of wild type and key mutant enzymes to study the mechanism of the biotin carboxylase domain where MgATP and bicarbonate carboxylate biotin. Further X-ray structures will be obtained of mutants and with bound reactants other than MgATP. 2) Use kinetic studies of wild type and key mutant enzymes to study the mechanism of the carboxyl transferase domain where carboxybiotin converts pyruvate to oxaloacetate. Further X-ray structures will be obtained of mutants and with bound analogs of pyruvate.3) To clarify the role of acetyl-CoA as an allosteric activator, X-ray crystal structures will be determined in its absence as well as its presence. Structures will be determined with enzymes from sources where acetyl-CoA is not an activator. Structures will also be determined in the presence of aspartate, an allosteric inhibitor, to determine where it binds and how it affects the structural changes caused by acetyl-CoA.4) Investigation of the interdomain movement of biotin and carboxybiotin will be carried out using 1D and 2D NMR. [1-15N]-biotin and the methyl and acetyl analogs will be covalently attached to the enzyme using biotin ligase and a biotin auxotroph system. This label will allow us to probe the location and to what extent biotin is present in each domain.5) Carboxyphosphate will be synthesized by saturating a solution of tris[tetrabutylammonium] phosphate in dimethyl formamide with CO2. A small amount of this solution will be mixed in a stopped flow apparatus with enzyme, Mg2+, ADP and buffer and the resulting mixture analyzed for ATP formation with a firefly luciferase assay. If ATP is formed, this will establish carboxyphosphate as an intermediate in the reaction.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 该项目的目的是使用我们最近解决的全长生物素丙酮酸羧化酶全酶的结构来确定这种重要的代谢酶的催化机理的细节。 我们的具体目的是：1）使用野生型和关键突变酶的动力学研究研究MGATP和碳酸氢盐羧酸盐生物素的生物素羧化酶结构域的机制。 将获得进一步的X射线结构，除MGATP以外的突变体和结合反应物。 2）使用野生型和关键突变酶的动力学研究来研究羧基转移酶结构域的机理，其中羧酸蛋白将丙酮酸转化为草乙酸。 将获得进一步的X射线结构，并具有丙酮酸的结合类似物。3）为了阐明乙酰辅酶A作为变构激活剂的作用，将在不存在及其存在下确定X射线晶体结构。 结构将由来自乙酰辅酶A不是激活剂的来源的酶确定。 结构还将在天冬氨酸存在的存在（一种变构抑制剂）的存在下确定，以确定其结合的位置以及如何影响乙酰基-COA引起的结构变化。4）研究将使用1D和2D NMR来研究生物素和羧二比蛋白的域间运动。 [1-15N] - 生物素和甲基和乙酰类似物将使用生物素连接酶和生物素型核酸菌属系统共价附着在酶上。 该标签将使我们能够探测每个结构域中生物素的位置。5）羧磷酸盐将通过饱和二甲基甲基酰胺中CO2的Tris [Tetrabutylymonium]磷酸的溶液来合成。 少量该溶液将与酶，MG2+，ADP和缓冲液一起在停止的流动设备中混合，并用萤火虫荧光素酶测定法分析了ATP形成的混合物。 如果形成ATP，这将在反应中建立羧磷酸盐作为中间体。该项目的目的是使用我们最近解决的全长含含生物素的丙酮酸羧化酶全酶的结构，以确定这种重要的代谢酶的催化机制的详细信息。 我们的具体目的是：1）使用野生型和关键突变酶的动力学研究研究MGATP和碳酸氢盐羧酸盐生物素的生物素羧化酶结构域的机制。 将获得进一步的X射线结构，除MGATP以外的突变体和结合反应物。 2）使用野生型和关键突变酶的动力学研究来研究羧基转移酶结构域的机理，其中羧酸蛋白将丙酮酸转化为草乙酸。 将获得进一步的X射线结构，并具有丙酮酸的结合类似物。3）为了阐明乙酰辅酶A作为变构激活剂的作用，将在不存在及其存在下确定X射线晶体结构。 结构将由来自乙酰辅酶A不是激活剂的来源的酶确定。 结构还将在天冬氨酸存在的存在（一种变构抑制剂）的存在下确定，以确定其结合的位置以及如何影响乙酰基-COA引起的结构变化。4）研究将使用1D和2D NMR来研究生物素和羧二比蛋白的域间运动。 [1-15N] - 生物素和甲基和乙酰类似物将使用生物素连接酶和生物素型核酸菌属系统共价附着在酶上。 该标签将使我们能够探测每个结构域中生物素的位置。5）羧磷酸盐将通过饱和二甲基甲基酰胺中CO2的Tris [Tetrabutylymonium]磷酸的溶液来合成。 少量该溶液将与酶，MG2+，ADP和缓冲液一起在停止的流动设备中混合，并用萤火虫荧光素酶测定法分析了ATP形成的混合物。 如果形成ATP，这将在反应中建立羧磷酸盐作为中间体。