TRANSCARBOXYLASE 13S STRUCTURE AND CO2 INTERMEDIATES

转羧酶 13S 结构和 CO2 中间体

• 批准号: 2388065
• 负责人: PAUL R CAREY
• 金额: $ 26.05万
• 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/2388065
• 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：含生物素的酶存在于所有生物体中