The structure and function of pyruvate carboxylase

描述(申请人提供)：本项目的目的是利用我们最近解决的含有生物素的全长丙酮酸羧基酶的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生物素催化丙酮酸转化为草酰乙酸酯过程中的作用机制。进一步的X射线结构将得到突变体和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，将在没有和存在的情况下测定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。还将在天冬氨酸(一种变构抑制剂)存在的情况下确定其结构，以确定它在哪里结合，以及它如何影响乙酰辅酶A引起的结构变化。乙酰辅酶A结合与催化循环中的步骤之间的关系将使用荧光乙酰辅酶A类似物来探索。4)用一维和二维核磁共振研究生物素和羧基生物素的结构域间运动。[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位置，并确定生物素在每个区域中存在的程度。与结构域间运动相关的构象变化的动力学将通过将色氨酸和香豆基荧光氨基酸类似物掺入BCCP和CT结构域的特定位置来研究。根据亚基对的构象不同，这些残基的接近程度将通过使用停流法的荧光共振能量转移来测量。5)将磷酸三[四丁基铵]溶液与二氧化碳浸泡在二甲基甲酰胺中，即可合成羧基磷酸盐。将少量的这种溶液与酶、Mg2、ADP和缓冲液混合在停流装置中，得到的混合物用萤火虫荧光素酶试验分析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。与公众健康相关：该项目的目标是确定丙酮酸羧基酶的结构和功能，丙酮酸羧基酶是肝脏、肾脏、大脑、胰岛、乳腺和脂肪组织等主要器官正常功能所必需的酶。缺乏会导致正常血糖水平的失控，严重而广泛的代谢紊乱，包括大脑功能异常和过早死亡，过度表达与2型糖尿病和肥胖症有关。研究中使用的工具是X射线结晶学、关键突变体的创建以及通过稳态和稳态前动力学来表征它们。

DESCRIPTION (provided by applicant): 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 determine 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. PUBLIC HEALTH RELEVANCE: The goal of this project is to determine the structure and function of pyruvate carboxylase, an essential enzyme in the normal function of major organs such as liver, kidney, brain, pancreatic islets, mammary gland and adipose tissue. Deficiency leads to loss of control of normal blood sugar levels, severe and widespread metabolic disturbances, including abnormal brain function and early death, and overexpression is associated with type 2 diabetes and obesity. The tools used in the study are X-ray crystallography, creation of key mutants and their characterization by steady state and pre-steady state kinetics.