The structure and function of pyruvate carboxylase

摘要/摘要 本项目的目的是利用含生物素丙酮酸的全长结构 羧化酶，我们最近已经解决，以确定催化机制的细节 这一重要的代谢酶。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 生物素羧化酶结构域，其中MgATP和碳酸氢盐羧化生物素。另外的x射线 将获得突变体的结构，并与MgATP以外的结合反应物。 2)使用野生型和关键突变酶的动力学研究来研究 羧基生物素将丙酮酸转化为草酰乙酸的羧基转移酶结构域。进一步 将获得突变体和结合丙酮酸类似物的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，X射线晶体结构将 它的存在和它的存在一样重要。结构将根据 来自乙酰辅酶A不是激活剂的来源的酶。还将确定结构 在天冬氨酸，一种变构抑制剂的存在下，以确定它在哪里结合以及如何结合。 影响乙酰辅酶A引起的结构变化。乙酰辅酶A与 结合和步骤，在催化循环相对于假定的一半的网站的反应性 将使用荧光乙酰辅酶A类似物进行探测。 4)研究生物素和羧基生物素的结构域间运动 使用1D和2D NMR。[1- 15 N]-生物素和甲基和乙酰基类似物将共价结合。 使用生物素连接酶和生物素营养缺陷型系统连接到酶上。这个标签将允许 我们来探测生物素在每个结构域中的位置和存在程度。的动力学 与结构域间运动相关的构象变化将通过 色氨酸和香豆酰荧光氨基酸类似物掺入特定位点 在BCCP和CT领域。这些残基的接近程度，根据不同的 将通过荧光共振能量测量亚基对的构象 转移，使用停流方法。 5)羧基磷酸盐将通过饱和以下物质的溶液合成： 磷酸三[四丁基铵]在二甲基甲酰胺中与CO2的混合物。少量这种 溶液将在停流装置中与酶、Mg 2+、ADP和缓冲液混合， 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， 这将建立磷酸根作为反应中的中间体。

Summary/Abstract The purpose of this project is to use the structure of the full length biotin-containing pyruvate carboxylase 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 carboxytransferase 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. The relationship between acetyl CoA binding and steps in the catalytic cycle with respect to the postulated half-of-the sites reactivity will be probed using fluorescent acetyl CoA analogues. 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 the biotin is present in each domain. The kinetics of conformational changes associated with interdomain movements will be investigated by incorporation of a tryptophan and a coumaryl fluorescent amino acid analogue into specific sites in the BCCP and CT domains. The proximity of these residues, which varies according to the conformation of the pair of subunits, will be measured by fluorescence resonance energy transfer, using stopped-flow methods. 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.