SUBUNITS AND QUATERNARY STRUCTURE OF TRANSCARBOXYLASE

转羧酶的亚基和四级结构

• 批准号: 3298713
• 负责人: DAVID SAMOLS
• 金额: $ 35.78万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-09-01 至 1993-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3298713
• 关键词: Escherichia coli; Propionibacterium; X ray crystallography; biotin; chemical binding; chemical group; chemical structure function; conformation; electron microscopy; enzyme mechanism; enzyme reconstitution; enzyme structure; enzyme substrate; high performance liquid chromatography; ligase; lysine; molecular cloning; nuclear magnetic resonance spectroscopy; oligopeptides; point mutation; protein engineering; protein sequence; site directed mutagenesis

Transcarboxylase is a biotin-containing enzymes from the bacterium, Propionibacterium shermanii. It is but one of many biotin enzymes which are widespread in organisms. They occur in mammals and catalyze important steps in gluconeogenesis, lipogenesis and amino acid metaboslim. In procaryotes, they have been found to generate proton gradients across members. A deficiency of these enzymes in genetic disorders of infants is often fatal. Transcarboxylase is the best characterized of all the biotin enzymes. It is made up of 3 different types of subunits, each of which has a different catalytic function, which is required in the overall reaction. These subunits can be isolated and their individual activity measured in the partial reactions. In addition, the intact active enzyme can be reconstituted from the individual subunits and is fully active. Each subunit has been cloned and its amino acid sequence determined. With the clones as tools, by site-directed mutagenesis, the structures will be altered at specific sites of the individual subunits. The overall aim is to dissect the structure of each subunit and determine the relationship of its structure to its catalytic function in relation to the action of the intact enzyme. The action of biotin is as the carboxyl carrier in biotin enzymes and in all biotin enzymes there is a conserved Val/Ala Met Bct Met surrounding the biotin (Bct is biotinyl lysine). This conservation during millions of years of evolution almost certainly indicates this sequence is essential for the catalysis of biotin enzymes in general, or that it provides the signal that informs the synthetase which attaches the biotin to the lysine, that this is the particular lysine that is to be biotinated posttranslationally. One aim will be to make changes at this conserved site and others of the biotinyl subunit to determine the exact requirement for the biotin to act as a carboxly carrier and to determine what directs the synthetase to the lysine that is to be biotinated. Site-directed mutagenesis will also be done to determine the functional domains of the other two subunits. To provide further information relating to structure and function, one aim will be to determine the three-dimensional structure of the subunits and mutants of the subunits by X-ray and electron microscopy of their crystals and thus correlate the changes in tertiary structure with changes in functions observed by site- directed mutagenesis.

转羧化酶是一种含生物素的酶类