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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 该项目的目的是利用我们最近解析的全长含生物素丙酮酸羧化酶全酶的结构来确定这种重要代谢酶的催化机制的细节。 我们的具体目标是：1）利用野生型和关键突变酶的动力学研究来研究生物素羧化酶结构域（其中MgATP和碳酸氢盐生物素）的机制。 将获得突变体和除 MgATP 之外的结合反应物的进一步 X 射线结构。 2) 利用野生型和关键突变酶的动力学研究来研究羧基生物素将丙酮酸转化为草酰乙酸的羧基转移酶结构域的机制。 将获得突变体和结合丙酮酸类似物的进一步X射线结构。3) 为了阐明乙酰辅酶A作为变构激活剂的作用，将确定其不存在和存在的情况下的X射线晶体结构。 将使用来自乙酰辅酶A不是激活剂的来源的酶来确定结构。 还将在天冬氨酸（一种变构抑制剂）存在的情况下确定结构，以确定其结合位置以及它如何影响乙酰辅酶A引起的结构变化。4) 将使用 1D 对生物素和羧基生物素的域间运动进行研究和二维核磁共振。 [1-15N]-生物素以及甲基和乙酰基类似物将使用生物素连接酶和生物素营养缺陷型系统共价连接到酶上。 该标记将使我们能够探测每个结构域中生物素的位置和存在程度。5) 通过用 CO2 饱和磷酸三[四丁基铵]二甲基甲酰胺溶液来合成羧磷酸盐。 少量该溶液将在停流装置中与酶、Mg2+、ADP 和缓冲液混合，并使用萤火虫荧光素酶测定分析所得混合物的 ATP 形成。 如果形成 ATP，则将形成羧基磷酸盐作为反应中的中间体。该项目的目的是利用我们最近解决的全长含生物素丙酮酸羧化酶全酶的结构来确定催化的细节。这种重要的代谢酶的机制。 我们的具体目标是：1）利用野生型和关键突变酶的动力学研究来研究生物素羧化酶结构域（其中MgATP和碳酸氢盐生物素）的机制。 将获得突变体和除 MgATP 之外的结合反应物的进一步 X 射线结构。 2) 利用野生型和关键突变酶的动力学研究来研究羧基生物素将丙酮酸转化为草酰乙酸的羧基转移酶结构域的机制。 将获得突变体和结合丙酮酸类似物的进一步X射线结构。3) 为了阐明乙酰辅酶A作为变构激活剂的作用，将确定其不存在和存在的情况下的X射线晶体结构。 将使用来自乙酰辅酶A不是激活剂的来源的酶来确定结构。 还将在天冬氨酸（一种变构抑制剂）存在的情况下确定结构，以确定其结合位置以及它如何影响乙酰辅酶A引起的结构变化。4) 将使用 1D 对生物素和羧基生物素的域间运动进行研究和二维核磁共振。 [1-15N]-生物素以及甲基和乙酰基类似物将使用生物素连接酶和生物素营养缺陷型系统共价连接到酶上。 该标记将使我们能够探测每个结构域中生物素的位置和存在程度。5) 通过用 CO2 饱和磷酸三[四丁基铵]二甲基甲酰胺溶液来合成羧磷酸盐。 少量该溶液将在停流装置中与酶、Mg2+、ADP 和缓冲液混合，并使用萤火虫荧光素酶测定分析所得混合物的 ATP 形成。 如果形成 ATP，则会形成磷酸酯作为反应中的中间体。