The Role of Self-Processing in Galactose Oxidase

描述(由申请人提供)： 这个项目建议研究自我加工在真菌蛋白半乳糖氧化酶中的作用。在成熟的形式中，这种酶具有一种修饰的酪氨酸辅因子，它在酶将酒精底物氧化为相应醛的能力中起着至关重要的作用。辅因子的生物发生需要铜和氧气，并可能与另一个翻译后修饰相连，该修饰导致半乳糖氧化酶1.7 kDa的前体序列被切割。半乳糖氧化酶自处理前后的晶体结构显示了明显的构象变化，这些变化可能会影响铜的配位球及其氧化态。氧被认为是前序列裂解的变构效应器，而前序列裂解又负责构象变化，使生物发生。这些假设可以通过表征自我加工的产物并分别测量前序列裂解和辅因子生物发生的速率来检验。通过添加试剂或突变对自我处理的扰动将被用来确定潜在的速率决定步骤，并开发一种将前序列切割与辅因子生物发生联系起来的综合机制。

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.