KINETIC INVESTIGATION OF PYRUVATE CARBOXYLASE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 本项目的目的是利用我们最近解决的含有生物素的丙酮酸羧化酶全长结构来确定这一重要代谢酶的催化机理的细节。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的机制，其中镁-三磷酸腺苷和碳酸氢钠羧化生物素。进一步的X射线结构将获得突变体和与除镁三磷酸腺苷以外的结合反应物。2)利用野生型和关键突变酶的动力学研究，研究羧基转移酶结构域在羧基转移酶结构域中转化丙酮酸为草酰乙酸酯的机理。3)为了阐明乙酰辅酶A作为变构激活剂的作用，将在没有和存在的情况下确定X射线晶体结构。结构将用来自乙酰辅酶A不是活化剂的来源的酶来确定。也将在天冬氨酸(一种变构抑制剂)的存在下确定结构，以确定它与哪里结合以及它如何影响乙酰辅酶A引起的结构变化。4)将使用一维和二维核磁共振对生物素和羧基生物素的结构域间运动进行研究。[1-15N]-生物素与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。这个标签将允许我们探测每个区域中生物素的位置和存在的程度。5)通过将磷酸三[四丁基铵]溶液浸泡在二甲基甲酰胺和二氧化碳中合成羧基磷酸盐。少量的这种溶液将在停流装置中与酶、镁、ADP和缓冲液混合，得到的混合物将用萤火虫荧光素酶分析ATP的形成。如果形成ATP，这将建立羧磷酸作为反应的中间体。本项目的目的是利用我们最近解决的含有生物素的丙酮酸羧化酶全长结构来确定这一重要代谢酶的催化机理的细节。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的机制，其中镁-三磷酸腺苷和碳酸氢钠羧化生物素。进一步的X射线结构将获得突变体和与除镁三磷酸腺苷以外的结合反应物。2)利用野生型和关键突变酶的动力学研究，研究羧基转移酶结构域在羧基转移酶结构域中转化丙酮酸为草酰乙酸酯的机理。3)为了阐明乙酰辅酶A作为变构激活剂的作用，将在没有和存在的情况下确定X射线晶体结构。结构将用来自乙酰辅酶A不是活化剂的来源的酶来确定。也将在天冬氨酸(一种变构抑制剂)的存在下确定结构，以确定它与哪里结合以及它如何影响乙酰辅酶A引起的结构变化。4)将使用一维和二维核磁共振对生物素和羧基生物素的结构域间运动进行研究。[1-15N]-生物素与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。这个标签将允许我们探测每个区域中生物素的位置和存在的程度。5)通过将磷酸三[四丁基铵]溶液浸泡在二甲基甲酰胺和二氧化碳中合成羧基磷酸盐。少量的这种溶液将在停流装置中与酶、镁、ADP和缓冲液混合，得到的混合物将用萤火虫荧光素酶分析ATP的形成。如果生成了三磷酸腺苷，这将在反应中建立作为中间体的羧基磷酸。

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.