TRANSCARBOXYLASE 13S STRUCTURE AND CO2 INTERMEDIATES

描述：含有生物素的酶存在于所有生物体和 催化代谢途径中的关键步骤。在人类中，这些缺陷 酶可能会产生严重的或灾难性的后果。尽管 持续的机械和医学兴趣，对细节知之甚少 这一密切相关的蛋白质家族的结构和化学。这个 这项研究的战略目标是定义1.3的原子细节 转羧酶(TC)及其中间体的S亚基 羧酸基与生物素辅因子瞬时结合。 多维异核核磁共振将用于解决结构 溶液中的全息蛋白和载脂蛋白-1.3的S蛋白。这些蛋白质将被标记 15N和/或13C。蛋白质上的生物素辅助因子的性质， 与其作为羧酸受体、转运体和 供体，将通过拉曼光谱和FTIR差示光谱以及 核磁共振。 羧基-生物素络合物的结构和电子分布 1.3.S亚基也将由振动和 核磁共振波谱。此外，蛋白质S结构的变化 生物素的羧化作用将通过核磁共振进行研究。 最近的FTIR数据表明，在二氧化碳部分离开生物素环后 在1.3S上，通过自发脱羧基，它仍然与 到目前为止，蛋白质是一种尚未确定特征的复合体。这个建筑群将是 使用病毒光谱技术和核磁共振进行鉴定，此外 涉及旨在使用CH2N2以稳定形式捕获络合物的实验。 作为催化能力的测试，新的络合物将与 丙酮酸在5S亚基的存在下，看看是否形成草酸乙酯。 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分异构体将与量子相结合 为解释以下数据提供基础的机械计算 1.3s上的生物素和羧基生物素。

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.