Covalent Phosphorus Residues in Xanthine Oxidizing Enzymes

黄嘌呤氧化酶中的共价磷残基

• 批准号: 9317614
• 负责人: Dale Edmondson
• 金额: $ 4.7万
• 依托单位: Emory University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-01-01 至 1995-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9317614&HistoricalAwards=false
• 关键词: Covalent; Phosphorus; Residues; Xanthine; Oxidizing

Edmondson 9317614 This proposal is to investigate the possible catalytic role of a phosphoseryl residue in the catalytic mechanism of xanthine hydroxlation to uric acid by xanthine oxidase/dehydrogenase. Previous studies have shown the phosphoseryl oxygens to be labelled with H2180 in milk xanthine oxidase on substrate turnover. This line of investigation will be expanded to determine if the 190 labelled phosphoseryl residue is transferred to the substrate to form 190 labelled uric acid when the reaction is carried out in H2160. The phosphoseryl peptides will be isolated from chicken liver xanthine dehydrogenase and from milk xanthine oxidase and sequenced. The peptide sequence around these residues will then be located in the known sequence of the chicken enzyme and the known sequences of the rat or Drosophila enzymes to determine if they are located about the Molybdenum-cofactor binding domain. These experiments will critically test the feasibility of the hypothesis of a catalytic involvement of the phosphoseryl residues in this class of molybdoenzymes. %%% This project addresses a hypothesis of a potentially new function of phosphorylated amino acid residues in the detailed molecular mechanism of molybdoenzyme catalysis. Peptide sequence analysis of phosphopeptides will be isolated from proteolytic digests of chicken liver and bovine milk xanthine oxidizing enzymes, their amino acid sequences determined, and their respective locations in the respective amino acid sequences of known xanthine oxidizing enzymes determined. This information will determine if they are located near the domains of the enzyme thought to contain the active site molybdenum cofactor. The pathway for H2160 incorporation into the product (uric acid) of the enzyme catalyzed reaction will be followed by monitoring 180 labelling of the phosphoseryl phosphate residue and the uric acid product by mass spectrometry. These experiments should provide definitive evidence as to whether the phosphoseryl residue in xanthine oxidizing enzymes functions in a catalytic role. If this hypothesis proves correct, it would be the first demonstration of such a function for enzyme phosphorylation. ***

Edmondson 9317614该建议是研究磷酸丝氨酰残基在黄嘌呤氧化酶/脱氢酶将黄嘌呤羟基化为尿酸的催化机制中可能的催化作用。 以往的研究表明，磷酸丝氨酰氧被标记与H2180在牛奶黄嘌呤氧化酶的底物周转。 这一研究路线将扩大，以确定当在H2160中进行反应时，190标记的磷酸丝氨酰残基是否转移到底物上形成190标记的尿酸。 将从鸡肝黄嘌呤脱氢酶和牛奶黄嘌呤氧化酶中分离磷酸丝氨酰肽并进行测序。 然后将这些残基周围的肽序列定位在鸡酶的已知序列和大鼠或果蝇酶的已知序列中，以确定它们是否位于钼辅因子结合结构域周围。 这些实验将严格检验磷酸丝氨酰残基催化参与这类酶的假设的可行性。 本项目提出了一个假设，即磷酸化氨基酸残基在辅酶催化的详细分子机制中具有潜在的新功能。 磷酸肽的肽序列分析将从鸡肝和牛乳黄嘌呤氧化酶的蛋白水解酶中分离，测定它们的氨基酸序列，并测定它们在已知黄嘌呤氧化酶的相应氨基酸序列中的相应位置。 这些信息将确定它们是否位于被认为含有活性位点钼辅因子的酶的结构域附近。 H2160掺入酶催化反应产物（尿酸）的途径将通过质谱法监测磷酸丝氨酰磷酸残基和尿酸产物的标记。 这些实验应提供明确的证据，以磷酸丝氨酰残基在黄嘌呤氧化酶功能的催化作用。 如果这一假设被证明是正确的，这将是首次证明酶磷酸化的这种功能。 ***