The Role of Self-Processing in Galactose Oxidase

自我加工在半乳糖氧化酶中的作用

• 批准号: 6583605
• 负责人: KRISTI J HUMPHREYS
• 金额: $ 4.16万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-06-30 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6583605
• 关键词: SDS polyacrylamide gel electrophoresis; chemical binding; chemical cleavage; chemical kinetics; cofactor; conformation; copper; enzyme activity; enzyme structure; free radical oxygen; galactose oxidase; high performance liquid chromatography; oxygen consumption; postdoctoral investigator; site directed mutagenesis; ultraviolet spectrometry

DESCRIPTION (provided by applicant): This project proposes to investigate the role of self-processing in the fungal protein, galactose oxidase. In the mature form, this enzyme possesses a modified tyrosine cofactor that plays a crucial role in the enzyme's ability to oxidize alcoholic substrates to their corresponding aldehydes. Biogenesis of the cofactor requires both copper and dioxygen and may be connected to another post-translational modification that results in cleavage of a 1.7 kDa pro-sequence from galactose oxidase. Crystal structures of galactose oxidase before and after self-processing reveal significant conformational changes that may influence the copper coordination sphere and its oxidation state. Dioxygen is proposed to act as an allosteric effector of pro-sequence cleavage, which in turn is responsible for a conformational change allowing biogenesis to occur. These suppositions can be tested by characterizing the products of self-processing and separately measuring the rates of both pro-sequence cleavage and cofactor biogenesis. Perturbations to self-processing through added reagents or mutagenesis will be used to identify potentially rate-determining steps and develop a comprehensive mechanism that links pro-sequence cleavage to cofactor biogenesis.

描述（由申请人提供）： 本计画拟探讨真菌蛋白质半乳糖氧化酶之自我加工作用。在成熟形式中，该酶具有修饰的酪氨酸辅因子，其在酶将醇底物氧化成其相应醛的能力中起关键作用。辅因子的生物发生需要铜和分子氧，并且可以连接到另一个翻译后修饰，其导致从半乳糖氧化酶切割1.7 kDa的前序列。半乳糖氧化酶自处理前后的晶体结构揭示了可能影响铜配位球及其氧化态的显着构象变化。分子氧被认为是前序列裂解的变构效应物，而前序列裂解又导致构象变化，从而使生物发生。这些假设可以通过表征自我加工的产物和分别测量前序列切割和辅因子生物合成的速率来测试。通过添加试剂或诱变对自处理的扰动将用于识别潜在的速率决定步骤，并开发将前序列切割与辅因子生物发生联系起来的综合机制。