TRANSCARBOXYLASE 13S STRUCTURE AND CO2 INTERMEDIATES

转羧酶 13S 结构和 CO2 中间体

• 批准号: 2734247
• 负责人: PAUL R CAREY
• 金额: $ 21.97万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-15 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2734247
• 关键词: Raman spectrometry; aminoacid metabolism; bicarbonates; biotin; carbon dioxide; carboxyltransferase /carbamoyltransferase; chemical kinetics; infrared spectrometry; interferometry; nuclear magnetic resonance spectroscopy; phosphates; protein structure function; quantum chemistry

DESCRIPTION: Biotin containing enzymes are found in all organisms and catalyze key steps in metabolic pathways. In humans, deficiencies in these enzymes can have serious, or catastrophic, consequences. In spite of continuing mechanistic and medical interest, little is known of the detailed structure and chemistry of this closely related family of proteins. The strategic goals of this research are to define the atomic details of the 1.3 S subunit of transcarboxylase (TC) and of its intermediate which has a carboxylate group transiently bound to the biotin cofactor. Multidimensional heteronuclear NMR will be used to solve the structures of the holo- and apo- 1.3 S protein in solution. The proteins will be labeled with 15N and/or 13C. The properties of the biotin co-factor on the protein, which relate to its function as a carboxylate acceptor, transporter and donor, will be elucidated by Raman and FTIR difference spectroscopies and by NMR. The structure and electron distribution in the carboxy-biotin complex on the 1.3 S subunit will be determined also by a combination of vibrational and NMR spectroscopies. In addition, changes in the protein s structure upon biotin carboxylation will be probed by NMR. Recent FTIR data indicate that after the CO2 moiety leaves the biotin ring on 1.3S via spontaneous decarboxylation, it remains associated witht he protein as an, as yet, uncharacterized complex. The complex will be identified using virbational spectroscopic techniques and NMR, in addition to experiments aimed at trapping the complex in a stable form using CH2N2. As a test of catalytic competence, the novel complex will be reacted with pyruvate in the presence of the 5S subunit to see if oxalacetate is formed. Vibrational spectroscopic analysis of biotin model compounds, of carboxy-biotin compounds and their isotopomers will be combined with quantum mechanical calculations to provide a basis for interpreting the data for biotin and carboxy-biotin on 1.3S.

描述：在所有生物体中发现含有生物素的酶， 催化代谢途径中的关键步骤。 在人类中，这些缺陷 酶可能会产生严重或灾难性的后果。 尽管 持续的机械和医学兴趣，很少有人知道详细的 这个密切相关的蛋白质家族的结构和化学。 的 这项研究的战略目标是确定1.3的原子细节， 转羧酶（TC）及其中间体的S亚基， 羧酸基团与生物素辅因子瞬时结合。 多维异谱NMR将用于解决结构的 溶液中的holo-和apo-1.3S蛋白。 蛋白质会被标记 15 N和/或13 C。 蛋白质上生物素辅因子的性质， 其涉及其作为羧酸受体、转运蛋白和 供体，将通过拉曼和FTIR差谱法以及 匪r 研究了羧基-生物素复合物在不同温度下的结构和电子分布。 1.3 S亚基也将由振动和 NMR光谱。 此外，蛋白质结构的变化， 生物素羧化将通过NMR探测。 最近的FTIR数据表明，CO2部分离开生物素环后， 通过自发脱羧作用在1.3S上，它仍然与 蛋白质作为一个，迄今为止，未表征的复合物。 该综合体将是 使用振动光谱技术和NMR鉴定，此外， 涉及旨在使用CH2N2以稳定形式捕获复合物的实验。 作为催化能力的测试，新的络合物将与 在5S亚基的存在下，观察是否形成了丙酮酸。 生物素模型化合物的振动光谱分析 羧基-生物素化合物及其同位素异构体将与量子 机械计算，以提供解释数据的基础， 生物素和羧基生物素。