KINETIC INVESTIGATION OF PYRUVATE CARBOXYLASE

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 本项目的目的是利用我们最近解决的全长含生物素的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)使用野生型和关键突变酶的动力学研究来研究MgATP和碳酸氢盐羧酸生物素的生物素羧化酶结构域的机制。 进一步的X射线结构将获得突变体和结合反应物以外的MgATP。（二） 使用野生型和关键突变酶的动力学研究，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。 进一步的X射线结构将获得突变体和结合的类似物。3）为了阐明乙酰辅酶A作为变构激活剂的作用，X射线晶体结构将在其存在和不存在的情况下进行测定。 将用来自乙酰辅酶A不是激活剂的来源的酶测定结构。 还将在天冬氨酸（一种变构抑制剂）的存在下测定结构，以确定其结合的位置以及其如何影响由乙酰辅酶A引起的结构变化。4）将使用1D和2D NMR进行生物素和羧基生物素的结构域间运动的研究。 使用生物素连接酶和生物素营养缺陷型系统将[1- 15 N]-生物素和甲基和乙酰基类似物共价连接到酶上。 该标记将使我们能够探测生物素在每个结构域中的位置和存在程度。5）将通过用CO2饱和磷酸三[四丁基铵]在二甲基甲酰胺中的溶液来合成羧基磷酸盐。 将少量该溶液在停流装置中与酶、Mg 2+、ADP和缓冲液混合，并用萤火虫荧光素酶测定法分析所得混合物的ATP形成。 如果形成ATP，这将建立作为反应中间体的羧基磷酸。本项目的目的是利用我们最近解决的全长含生物素的丙酮酸羧化酶全酶的结构，以确定这一重要代谢酶的催化机制的细节。 我们的具体目标是： 1)使用野生型和关键突变酶的动力学研究来研究MgATP和碳酸氢盐羧酸生物素的生物素羧化酶结构域的机制。 进一步的X射线结构将获得突变体和结合反应物以外的MgATP。（二） 使用野生型和关键突变酶的动力学研究，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。 进一步的X射线结构将获得突变体和结合的类似物。3）为了阐明乙酰辅酶A作为变构激活剂的作用，X射线晶体结构将在其存在和不存在的情况下进行测定。 将用来自乙酰辅酶A不是激活剂的来源的酶测定结构。 还将在天冬氨酸（一种变构抑制剂）的存在下测定结构，以确定其结合的位置以及其如何影响由乙酰辅酶A引起的结构变化。4）将使用1D和2D NMR进行生物素和羧基生物素的结构域间运动的研究。 使用生物素连接酶和生物素营养缺陷型系统将[1- 15 N]-生物素和甲基和乙酰基类似物共价连接到酶上。 该标记将使我们能够探测生物素在每个结构域中的位置和存在程度。5）将通过用CO2饱和磷酸三[四丁基铵]在二甲基甲酰胺中的溶液来合成羧基磷酸盐。 将少量该溶液在停流装置中与酶、Mg 2+、ADP和缓冲液混合，并用萤火虫荧光素酶测定法分析所得混合物的ATP形成。 如果形成ATP，这将建立羧基磷酸作为反应的中间体。

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.