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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.

总结/摘要本项目的目的是利用含生物素丙酮酸的全长结构羧化酶，我们最近已经解决，以确定催化机制的细节这一重要的代谢酶。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，其中MgATP和碳酸氢盐羧化生物素。另外的x射线将获得突变体的结构，并与MgATP以外的结合反应物。 2)使用野生型和关键突变酶的动力学研究来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。进一步将获得突变体和结合丙酮酸类似物的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，X射线晶体结构将它的存在和它的存在一样重要。结构将根据来自乙酰辅酶A不是激活剂的来源的酶。还将确定结构在天冬氨酸，一种变构抑制剂的存在下，以确定它在哪里结合以及如何结合。影响乙酰辅酶A引起的结构变化。乙酰辅酶A与结合和步骤，在催化循环相对于假定的一半的网站的反应性将使用荧光乙酰辅酶A类似物进行探测。 4)将进行生物素和羧基生物素结构域间运动的研究使用1D和2D NMR进行分析。[1- 15 N]-生物素和甲基和乙酰基类似物将共价结合。使用生物素连接酶和生物素营养缺陷型系统连接到酶上。这个标签将允许我们来探测生物素在每个结构域中的位置和存在程度。的动力学与结构域间运动相关的构象变化将通过色氨酸和香豆酰荧光氨基酸类似物掺入特定位点在BCCP和CT领域。这些残基的接近程度，根据不同的将通过荧光共振能量测量亚基对的构象转移，使用停流方法。 5)羧基磷酸盐将通过饱和以下物质的溶液合成：磷酸三[四丁基铵]在二甲基甲酰胺中与CO2的混合物。少量这种溶液将在停流装置中与酶、Mg 2+、ADP和缓冲液混合，使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，这将建立作为反应中间体的羧基磷酸盐。

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.

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

DOI：
10.1016/j.bbrc.2013.10.066
发表时间：
2013-11-15
期刊：
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
影响因子：
3.1
作者：
Lietzan, Adam D.;St Maurice, Martin
通讯作者：
St Maurice, Martin

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

My favorite pyruvate carboxylase.