Protein interactions in purine synthesis

嘌呤合成中的蛋白质相互作用

• 批准号: 7228423
• 负责人: TIMOTHY B LANGSTON
• 金额: $ 9.8万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-05-19 至 2007-05-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7228423
• 关键词: Active Sites; Affinity Chromatography; Amides; Amines; Anabolism; Antimetabolites; Binding; Biochemical; Catalysis; Complementary DNA; Complex; Cultured Cells; Deuterium; Digestion; Enzymatic Biochemistry; Enzymes; Epitopes; Eukaryota; Eukaryotic Cell; Folate; Gel; Growth and Development function; High Pressure Liquid Chromatography; Hydrogen; Hydroxymethyltransferases; Immunoprecipitation; In Vitro; Kinetics; Label; Ligase; Location; Mammalian Cell; Maps; Mass Spectrum Analysis; Mechanics; Metabolic Pathway; Molecular Conformation; Molecular Genetics; Mouse Cell Line; Mutate; Pathway interactions; Peptides; Phosphoribosylamine-glycine ligase; Phosphoribosylformylglycinamidine synthase; Process; Protein Biosynthesis; Protein Chemistry; Proteins; Proteolysis; Proteomics; Purines; Radioisotopes; Reaction; Recombinants; Research; Ribonucleotides; Site; Solvents; Surface; System; Techniques; Thinking; Ursidae Family; Vertebral column; base; crosslink; fascinate; genetic analysis; glycine amide; improved; in vivo; insight; interest; nano; programs; protein protein interaction; purine; reconstitution; research study; yeast two hybrid system

DESCRIPTION (provided by applicant): Multistep pathways are commonly thought to involve substrate channeling, whereby the product of one enzyme is delivered to the active site of another enzyme. We propose to study this phenomenon in the mammalian purine biosynthetic pathway using mass spectrometry and a combination of biochemical and molecular genetic analysis. The biosynthetic pathway in eukaryotes has been evolutionarily streamlined by fusing the enzymes of several steps into separate domains in one protein, GARS-AIRS-GART (GART). In the process of studying folate antimetabolites, this lab identified, characterized and purified the tri-functional enzyme. GART is not the only multifunctional enzyme in eukaryotic purine anabolism, but in regards to substrate channeling, GART is perhaps the most interesting. GART performs the 2nd GARS), 3rd (GART) and 5th (AIRS) steps of purine biosynthesis. The intermediate 4th step is performed by phosphoribosylformylglycinamidine synthetase (FGAMS), a separate monofunctional protein. We have recently isolated the cDNA of FGAMS from a mouse cell line. Here, we propose to purify FGAMS for kinetic studies on substrate channeling in vitro and to investigate protein/protein interactions in vivo. In order to examine substrate channeling, the main kinetic studies with FGAMS will involve radioisotope dilution studies performed in the presence of tri-functional GART. Product will be assessed for specific activity and the results will determine whether intermediate products have access to solvent and therefore whether channeling is a tight or leaky process. The hypothesis that the enzymes of purine biosynthesis exist as a large complex will be examined by expression in cell culture of tagged FGAMS and analysis by mass spectrometry of complexes formed during purine synthesis. Using the advantage of TAP (tandem affinity purification)-tagged FGAMS, we will isolate FGAMS with associated protein complexes. Following gel purification, in-gel digestion, and nano-HPLC, peptides wilt be sequenced by mass spectrometry. The results of these studies should give us clear insight into the mechanics of substrate channeling, a fundamentally important phenomenon whose exact mechanism has remained elusive.

描述（由申请人提供）：多步途径通常被认为涉及底物通道，由此一种酶的产物被递送至另一种酶的活性位点。 我们建议使用质谱法以及生化和分子遗传分析相结合的方法来研究哺乳动物嘌呤生物合成途径中的这种现象。通过将几个步骤的酶融合到一种蛋白质 GARS-AIRS-GART (GART) 的不同结构域中，真核生物的生物合成途径已在进化上得到简化。在研究叶酸抗代谢物的过程中，本实验室对三功能酶进行了鉴定、表征和纯化。 GART 并不是真核嘌呤合成代谢中唯一的多功能酶，但就底物通道而言，GART 可能是最有趣的。 GART 执行嘌呤生物合成的第二步（GARS）、第三步（GART）和第五步（AIRS）。中间第四步由磷酸核糖甲酰甘氨脒合成酶（FGAMS）（一种单独的单功能蛋白）执行。我们最近从小鼠细胞系中分离出了 FGAMS 的 cDNA。在这里，我们建议纯化 FGAMS 用于体外底物通道动力学研究，并研究体内蛋白质/蛋白质相互作用。为了检查底物通道，FGAMS 的主要动力学研究将涉及在三功能 GART 存在下进行的放射性同位素稀释研究。将评估产品的特定活性，结果将确定中间产品是否能够接触到溶剂，从而确定通道是紧密的还是泄漏的过程。嘌呤生物合成酶作为大复合物存在的假设将通过标记的 FGAMS 在细胞培养物中的表达以及对嘌呤合成过程中形成的复合物的质谱分析来检验。利用 TAP（串联亲和纯化）标记的 FGAMS 的优势，我们将分离 FGAMS 与相关的蛋白质复合物。经过凝胶纯化、凝胶内消化和纳米 HPLC 后，将通过质谱法对肽进行测序。这些研究的结果应该让我们清楚地了解底物沟道的机制，这是一种根本上重要的现象，其确切机制仍然难以捉摸。