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The structure and function of pyruvate carboxylase

丙酮酸羧化酶的结构和功能

基本信息

批准号：
8260543
负责人：
IVAN RAYMENT
金额：
$ 35.7万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-05-01 至 2014-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8260543
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&apos -carboxybiotin Non-Insulin-Dependent Diabetes Mellitus Obesity Organ Oxaloacetates Phosphorus Play Protons Pyruvate Pyruvate Carboxylase 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 abstracting 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 tetrabutylammonium tool

项目摘要

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.

摘要/文摘

项目成果

期刊论文数量（23）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Regulation of the structure and activity of pyruvate carboxylase by acetyl CoA.

DOI：
10.1016/j.abb.2011.11.015
发表时间：
2012-03-15
期刊：
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
影响因子：
3.9
作者：
Adina-Zada, Abdussalam;Zeczycki, Tonya N.;Attwood, Paul V.
通讯作者：
Attwood, Paul V.

Insight into the carboxyl transferase domain mechanism of pyruvate carboxylase from Rhizobium etli.

DOI：
10.1021/bi9003759
发表时间：
2009-05-26
期刊：
BIOCHEMISTRY
影响因子：
2.9
作者：
Zeczycki, Tonya N.;St Maurice, Martin;Jitrapakdee, Sarawut;Wallace, John C.;Attwood, Paul V.;Cleland, W. Wallace
通讯作者：
Cleland, W. Wallace

Insights into the carboxyltransferase reaction of pyruvate carboxylase from the structures of bound product and intermediate analogs.