The structure and function of pyruvate carboxylase

项目描述（由申请人提供）：本项目目的是利用我们最近解决的全长含生物素丙酮酸羧化酶的结构，确定这种重要代谢酶催化机制的细节。我们的具体目标是：1)利用野生型和关键突变型酶的动力学研究，研究MgATP和碳酸氢盐羧化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。2)利用野生型和关键突变型酶的动力学研究，研究羧生物素将丙酮酸转化为草酰乙酸的羧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丙酮酸类似物。3)为了阐明乙酰辅酶a作为变构活化剂的作用，将测定其存在和不存在时的x射线晶体结构。结构将由乙酰辅酶a不是活化剂的来源的酶来确定。在天冬氨酸（一种变构抑制剂）存在的情况下，还将确定其结构，以确定其结合的位置以及它如何影响乙酰辅酶a引起的结构变化。乙酰辅酶a结合与催化循环步骤之间的关系与假定的一半位点的反应性将使用荧光乙酰辅酶a类似物进行探测。4)利用1D和2D NMR研究生物素和羧基生物素的结构域间运动。[1-15N]-生物素及其甲基和乙酰类似物将通过生物素连接酶和生物素营养不良系统共价连接到酶上。这个标签将使我们能够探测位置并确定生物素在每个域中存在的程度。将色氨酸和香豆基荧光氨基酸类似物掺入BCCP和CT结构域的特定位点，研究与结构域间运动相关的构象变化动力学。这些残基的接近度，根据对亚基的构象而变化，将通过荧光共振能量转移来测量，使用停止流动的方法。5)将磷酸三[四丁基铵]溶液在二甲基甲酰胺中与二氧化碳饱和合成羧磷酸。将少量溶液与酶、Mg2+、ADP和缓冲液混合在停止流动装置中，用萤火虫荧光素酶测定分析所得混合物中ATP的形成。如果形成ATP，这将建立羧酸磷酸酯作为反应的中间产物。公共卫生相关性：该项目的目标是确定丙酮酸羧化酶的结构和功能，丙酮酸羧化酶是肝、肾、脑、胰岛、乳腺和脂肪组织等主要器官正常功能中必不可少的酶。缺乏导致正常血糖水平失控，严重和广泛的代谢紊乱，包括脑功能异常和早期死亡，过度表达与2型糖尿病和肥胖有关。研究中使用的工具是x射线晶体学，关键突变体的创建及其稳态和预稳态动力学的表征。

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.