STRUCTURES & MECHANISMS OF CITRATE ENZYMES

结构

• 批准号: 3224778
• 负责人: PAUL A SRERE
• 金额: $ 13.93万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-01-01 至 1994-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3224778
• 关键词: Escherichia coli; X ray crystallography; chemical structure function; citrate synthase; complementary DNA; conformation; enzyme mechanism; enzyme structure; genetic manipulation; molecular cloning; molecular site; point mutation; protein engineering; protein sequence; radiotracer; structural genes; swine

The goal of the research is to examine the structure and function of citrate synthase (CS) from pig and E. coli by using site-directed mutagenesis. Citrate synthase is an excellent choice to examine enzyme structure and function by using such molecular biological techniques. It is a key enzyme in aerobic energy production and metabolite interconversions. It is an important example of stereospecificity in enzyme reactions, and two distinct enzyme conformations participate during catalysis. In addition, pig citrate synthase (PCS) and E. coli citrate synthase (ECCS) have been crystallized and the three dimensional structure of PCS determined. Therefore, CS is an attractive enzyme to study by site-directed mutagenesis because of the possibility of obtaining the DNA, the protein crystals, and an in depth mechanism for the mammalian and bacterial forms of the enzyme. This additional comparative aspect between the CS from a eucaryote and a procaryote will enhance our ability to choose sites for mutagenesis, and also will explain the two different reaction mechanisms, protein structures, and regulatory behaviors of these divergently related proteins. To define in detail the reaction mechanism, structure, and biological function of CS, the cDNA encoding PCS will be isolated and sequenced. Site-directed mutagenesis of the PCS and ECCS DNAs will used to alter codons for catalytic and structural amino acid residues. The mutated and control DNAs will be expressed in vitro, and the synthesized proteins will be purified and crystallized. The partial enzyme reactions catalyzed by the control and mutated PCS and ECCS proteins will be determined and compared. The three dimensional structures of the control and mutated PCS and ECCS proteins will be compared to the known X-ray structure of PCS. In addition, the gene for an E. coli mutant that codes for the dimeric (eucaryotic) form of the enzyme will be isolated and sequenced. The amino acid sequence for the mutant ECCS will be compared to the PCS and non-mutant ECCS and may identify particualr amino acid residues that are important to subunit assembly or enzymatic function.

本研究的目的是研究其结构和功能

项目成果

Cooperation between enzyme and transporter in the inner mitochondrial membrane of yeast. Requirement for mitochondrial citrate synthase for citrate and malate transport in Saccharomyces cerevisiae.

酵母线粒体内膜中酶和转运蛋白之间的合作。

• 发表时间: 1994
• 期刊: The Journal of biological chemistry
• 作者: Sandor,A;Johnson,JH;Srere,PA
• 通讯作者: Srere,PA

Immunological mapping of fine molecular surface structures of citrate synthase enzymes from different cell types.

来自不同细胞类型的柠檬酸合酶的精细分子表面结构的免疫学图谱。

• DOI: 10.1002/jmr.300040206
• 发表时间: 1991
• 期刊: Journal of molecular recognition : JMR
• 作者: Nemeth,P;Small,WC;Evans,CT;Zhi,W;Persson,LO;Srere,PA
• 通讯作者: Srere,PA

17th Fritz Lipmann Lecture. Wanderings (wonderings) in metabolism.

第十七届弗里茨·李普曼讲座。

• 发表时间: 1993
• 期刊: Biological chemistry Hoppe-Seyler
• 作者: Srere,PA

Metabolic studies on Saccharomyces cerevisiae containing fused citrate synthase/malate dehydrogenase.

• DOI: 10.1021/bi00205a003
• 发表时间: 1994-10
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: C. Lindbladh;R. Brodeur;W. Small;G. Lilius;L. Bülow;K. Mosbach;P. Srere

The molecular physiology of citrate.

柠檬酸盐的分子生理学。

• DOI: 10.1016/b978-0-12-152833-1.50020-4
• 发表时间: 1992
• 期刊: Current topics in cellular regulation
• 作者: Srere,PA