The structure and function of pyruvate carboxylase

丙酮酸羧化酶的结构和功能

• 批准号: 7652146
• 负责人: WILLIAM Wallace CLELAND
• 金额: $ 37.68万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-05-01 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7652146
• 关键词: Acetyl Coenzyme A; Active Sites; Adipose tissue; Affect; Allosteric Regulation; Amino Acids; Apoenzymes; Aspartate; Aspergillus nidulans; Bicarbonates; Binding; Binding Sites; Biological Assay; Biotin; Biotin carboxylase; Blood Glucose; Brain; Buffers; Carbon Dioxide; Carboxyltransferases; Catalytic Domain; Cessation of life; Complex; Corynebacterium glutamicum; Decarboxylation; Engineering; Enzymes; Face; Family; Firefly Luciferases; Fluorescence Resonance Energy Transfer; Goals; Holoenzymes; Hybrids; Individual; Investigation; Islets of Langerhans; Isotopes; Kidney; Kinetics; Label; Length; Ligase; Liver; Location; Lysine; Mammary gland; Measures; Metabolic; Methods; Methylmalonyl-CoA carboxyltransferase; MgATP; Molecular Conformation; Movement; Mutagenesis; Mutation; N1' -carboxybiotin; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Organ; Oxaloacetates; Phosphorus; Play; Protons; Pyruvate; Pyruvate Carboxylase; Pyruvates; Reaction; Resolution; Roentgen Rays; Role; Sequence Alignment; Site; Site-Directed Mutagenesis; Solutions; Source; Staphylococcus aureus; Structure; System; Testing; Time; Translating; Tryptophan; Variant; X-Ray Crystallography; Yeasts; analog; biotin carboxyl carrier protein; carbonic acid, monoanhydride with phosphoric acid, ion(2-); carboxyl group; carboxylate; carboxylation; formamide; grasp; inhibitor/antagonist; inorganic phosphate; member; methyl group; microorganism; mutant; overexpression; public health relevance; tetrabutylammonium; tool

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.

描述（由申请人提供）：该项目的目的是使用含有生物素的全长丙酮酸羧化酶的结构，我们最近已经解决了该结构，以确定这种重要的代谢酶的催化机理的细节。我们的具体目的是：1）使用野生型和关键突变酶的动力学研究研究MGATP和碳酸氢盐羧酸盐生物素的生物素羧化酶结构域的机制。将获得进一步的X射线结构，除MGATP以外的突变体和结合反应物。 2）使用野生型和关键突变酶的动力学研究研究羧基二羧酸转化丙酮酸转化为草乙酸的羧基转移酶结构域的机制。将获得进一步的X射线结构，并具有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，将在其不存在的情况下确定X射线晶体结构。结构将由来自乙酰辅酶A不是激活剂的来源的酶确定。结构也将在天冬氨酸存在（一种变构抑制剂）的存在下确定，以确定其结合的位置以及如何影响乙酰-COA引起的结构变化。相对于假定的半位点反应性，乙酰基COA结合与催化循环中的步骤之间的关系将使用荧光乙酰基COA类似物进行探测。 4）将使用1D和2D NMR进行生物素和羧酸蛋白间运动的研究。 [1-15N] - 生物素和甲基和乙酰类似物将使用生物素连接酶和生物素型核酸菌属系统共价附着在酶上。该标签将使我们能够探测位置，并确定每个域中生物素存在的程度。与域间运动相关的构象变化的动力学将通过掺入色氨酸和COMARYL荧光氨基酸类似物的融合到BCCP和CT域中的特定位点。这些残基的接近度根据对亚基对的构象而变化，将通过荧光共振能量转移，使用停止流量方法来测量。 5）羧磷酸盐将通过饱和二甲基甲基酰胺的Tris溶液饱和，并通过二甲基二酰胺与CO2合成。少量该溶液将与酶，MG2+，ADP和缓冲液一起在停止的流动设备中混合，并用萤火虫荧光素酶测定法分析了ATP形成的混合物。如果形成ATP，这将在反应中建立羧磷酸盐作为中间体。公共卫生相关性：该项目的目的是确定丙酮酸羧化酶的结构和功能，这是主要器官（例如肝脏，肾脏，脑，脑，胰岛，乳腺和脂肪组织）正常功能的重要酶。缺乏会导致无法控制正常血糖水平，严重和广泛的代谢障碍，包括脑功能异常和早期死亡，并且过表达与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通过稳态和稳态态动力学的表征。