ID RESIDUE IN ACC OXIDASE ACTIVATION, TERMINAL ENZYME OF ETHYLENE BIOSYNTHESIS

ACC 氧化酶激活中的 ID 残留物，乙烯生物合成的末端酶

• 批准号: 6258826
• 负责人: HANS KENDE
• 金额: --
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-06-01 至 1999-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6258826
• 关键词: animal tissue; biological products; biomedical resource; blood; genetics; growth factor; proteins

Ethylene is a gaseous hormone that regulates a wide range of physiological responses in plants. The terminal enzyme in the biosynthetic pathway, 1-aminocyclopropane-1-carboxylate (ACC) oxidase, has recently been shown to be activated by CO2. Only one other enzyme has been shown to be activated by CO2 (ribulosebisphosphate carboxylase) via carbamate formation on the ?-amino group of a lysyl residue. By analogy, we are investigating whether a specific lysyl residue is modified by CO2 during activation. Because the carbamate is extremely labile, it will be necessary to form a stable methyl ester with the carbamate to allow proteolytic digestion while maintaining the carbamate. After proteolysis, the peptides will be analyzed by MALDI-MS to detect differences between the nonactivated and the CO2-activated enzyme. The enzyme has been purified after overexpression in E. coli. The purified form is estimated to be only 2-5% activated, and it can be fully activated by CO2. The mass spectrometry analysis will be crucial to determining which of the 26 absolutely conserved lysine residues is modified by CO2. We will then pursue site-directed mutagenesis and x-ray crystallography to confirm our results.

乙烯是一种气体激素，调节广泛的 植物的生理反应。 末端酶在 生物合成途径，1-氨基环丙烷-1-羧酸（ACC）氧化酶， 最近被证明是由二氧化碳激活的。 只有一种酶 已经显示出被CO2（核酮糖二磷酸）活化 羧化酶）通过氨基甲酸酯形成的？-赖氨酰的氨基 残余物 以此类推，我们正在研究一个特定的赖氨酰 残余物在活化期间被CO2改性。 因为氨基甲酸酯 是非常不稳定的，这将是必要的，以形成稳定的甲基， 与氨基甲酸酯酯化以允许蛋白水解消化， 保持氨基甲酸酯。 蛋白水解后，肽将被 通过MALDI-MS分析以检测未活化的 和二氧化碳活化酶 该酶已被纯化后， 在E.杆菌 据估计，纯化的形式只有 2-5%活化，可被CO2完全活化。 质量 光谱分析将是至关重要的，以确定哪些26 绝对保守的赖氨酸残基被CO2修饰。 然后我们将 进行定点突变和X射线晶体学来证实 我们的成果