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）：本项目的目的是使用我们最近解决的全长含生物素的丙酮酸羧化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生物素羧化酶结构域的机制，其中MgATP和碳酸氢盐羧化生物素。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)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。将获得突变体和结合丙酮酸类似物的进一步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，将在其存在和不存在的情况下测定X射线晶体结构。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）存在下测定结构，以确定其结合位置以及如何影响乙酰辅酶A引起的结构变化。乙酰辅酶A结合和步骤之间的关系，在催化循环相对于假定的一半的网站的反应性将探测使用荧光乙酰辅酶A类似物。4)将使用1D和2D NMR对生物素和羧基生物素的结构域间运动进行研究。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。这个标签将使我们能够探测位置，并确定生物素在每个结构域中的存在程度。通过将色氨酸和香豆酰荧光氨基酸类似物掺入BCCP和CT结构域中的特定位点，研究与结构域间运动相关的构象变化的动力学。这些残基的接近度根据亚基对的构象而变化，将使用停流方法通过荧光共振能量转移来测量。5)将通过用CO2使磷酸三[四丁基铵]在二甲基甲酰胺中的溶液饱和来合成羧基磷酸盐。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。公共卫生关系：本项目的目标是确定丙酮酸羧化酶的结构和功能，丙酮酸羧化酶是主要器官如肝、肾、脑、胰岛、乳腺和脂肪组织正常功能的必需酶。缺乏导致正常血糖水平失控，严重和广泛的代谢紊乱，包括脑功能异常和过早死亡，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表征。