REGULATION OF FATTY ACID BIOSYNTHESIS

脂肪酸生物合成的调节

• 批准号: 3285599
• 负责人: Charles O Rock
• 金额: $ 7.12万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-12-01 至 1988-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3285599
• 关键词: Escherichia coli; acetyl coA acetyltransferase; antibody; enzyme structure; fatty acid biosynthesis; gel electrophoresis; genetic mapping; growth /development; high performance liquid chromatography; membrane lipids; microorganism metabolism; structural genes; temperature sensitive mutant; tissue /cell culture

A considerable amount of metabolic energy is expended in the biogenesis of membrane lipid and organisms in general exert a high degree of control over the activity of this pathway. In Escherichia coli, the bulk of the available evidence indicates that the control point is at an early step in fatty acid biosynthesis, however, the definitive experimental identification of the rate-controlling enzyme has proved elusive and remains one of the major unanswered questions about bacterial physiology. The overall goal of this research program is to elucidate the mechanisms that regulate membrane lipid production and coordinate this process with growth and protein biosynthesis. An evaluation of the available data has led to the working hypothesis that control over the fatty acid biosynthetic rate is exerted at the level of chain initiation by modulation of acetyl-CoA:acyl carrier protein transacylase activity. This enzyme is ideally positioned in the pathway to control the number of fatty acids synthesized since the transfer of an acetyl group to acyl carrier protein yields the primer molecule required for starting new rounds of elongation. A combination of physiological, biochemical and genetic experimental approaches have been designed to test this working hypothesis against the alterative candidates for the regulatory enzyme. Acyl carrier protein-specific antibodies, high-pressure liquid chromatographic systems and a conformationally sensitive gel electrophoresis method will be used to directly determine the precursor pool present in the shortest supply in vivo. These measurements will either confirm that the supply of acetyl-acyl carrier protein is the rate-limiting factor or identify which of the other candidate enzymes catalyzes the slow step in fatty acid biosynthesis. As a further test of the hypothesis, acetyl transacylase will be purified and characterized to examine the biochemical mechanism of activity regulation in vitro. Temperature-sensitive acetyl transacylase mutants and strains exhibiting abnormal regulation of fatty acid biosynthesis will also be used to evaluate the role of this enzyme and to map the location of the transacylase structural gene on the E. coli chromosome.

大量的代谢能量消耗在生物发生过程中 膜脂和一般生物体对 该通路的活性。 在大肠杆菌中，大部分 现有证据表明，控制点正处于早期阶段 然而，脂肪酸生物合成是最终的实验 速率控制酶的鉴定已被证明是难以捉摸的 仍然是细菌生理学尚未解答的主要问题之一。 该研究计划的总体目标是阐明其机制 调节膜脂的产生并协调该过程 生长和蛋白质生物合成。 对现有数据的评估 导致了控制脂肪酸生物合成的工作假设 速率通过调节在链引发水平上发挥作用 乙酰辅酶A：酰基载体蛋白转酰基酶活性。 这种酶是 理想地位于控制脂肪酸数量的途径中 由于乙酰基转移至酰基载体蛋白而合成 产生开始新一轮延伸所需的引物分子。 生理、生化和遗传实验相结合 设计方法是为了测试这一工作假设 调节酶的替代候选者。 酰基载体 蛋白质特异性抗体、高压液相色谱系统 并采用构象敏感凝胶电泳方法 直接确定最短供应中存在的前体池 体内。 这些测量将确认供应 乙酰酰基载体蛋白是限速因子或确定哪个 其他候选酶催化脂肪酸的缓慢步骤 生物合成。 作为对假设的进一步检验，乙酰转酰基酶 将被纯化和表征以检查其生化机制 体外活性调节。 温度敏感乙酰基转酰基酶 脂肪酸调节异常的突变体和菌株 生物合成也将用于评估这种酶的作用并 绘制转酰基酶结构基因在大肠杆菌上的位置 染色体。