Structures & Redox Chemistry in Sulfinic Acid Reduction

结构

• 批准号: 7101960
• 负责人: W TODD LOWTHER
• 金额: $ 24.52万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7101960
• 关键词: X ray crystallography; active sites; catalyst; chemical kinetics; cofactor; enzyme complex; enzyme mechanism; enzyme structure; oxidation reduction reaction; oxidoreductase; peroxidases; site directed mutagenesis

DESCRIPTION (provided by applicant): The sulfinic acid reductase, sulfiredoxin (Srx), was recently identified as the enzyme in yeast responsible for the reduction of the sulfinic acid moiety (Cys-SO2-) within several oxidatively-inactivated peroxiredoxins (Prxs). This discovery shattered the dogma of the irreversibility of overoxidation for the Prx enzyme family. Moreover, the ATP- and Mg2+-dependent repair or "retroreduction" of the overoxidized Prxs may modulate the role of these enzymes as regulators of hydrogen peroxide-mediated intracellular signaling. Based on the analysis of the in vivo oxidation state for a variety of human Prxs, repair of different Prx isoforms by Srx appears to proceed at different rates, in spite of their high overall degree of sequence identity. Our recent structure determinations of human Srx in complex with either phosphate or ADP have revealed a new protein fold and a novel nucleotide binding motif. Other preliminary data have confirmed some aspects of the proposed scheme for yeast Srx action, although several surprises in our results (the isolation of a disulfide-bonded rather than thiosulfinate-linked intermediate, and the reduction of the Srx:Prx complex by glutathione rather than thioredoxin) have suggested that further investigation is required. The goals of this proposal are: to determine the crystal structures of human Srx in complex with cofactors and human Prxs (Aim 1); and to carry out steady-state and partial turnover experiments coupled with site-directed mutagenesis to elucidate the nature and reaction rates of intermediates, and to characterize residues critical to catalysis (Aims 2 & 3). These investigations will contribute significantly to our understanding of the molecular origins of sulfinic acid reductase action and the novel sulfur chemistry involved in this process.

描述（由申请人提供）：最近将硫酸还原酶（SRX）的硫酸还原酶硫酸氧化物（SRX）鉴定为酵母中的酶，负责在几种氧化灭菌的过氧化氧化氧化物（PRXS）中减少硫酸酸部分（CYS-SO2-）。这一发现破坏了PRX酶家族过度氧化的不可逆性的教条。此外，过度氧化PRX的ATP和MG2+依赖性修复或“逆转”可能会调节这些酶作为过氧化氢介导的细胞内信号传导的调节剂的作用。基于对各种人PRX的体内氧化态的分析，尽管其序列身份的总体程度很高，但SRX对不同PRX同工型的修复似乎以不同的速率进行。我们最近与磷酸盐或ADP复合物中人类SRX的结构测定显示了新的蛋白质折叠和一种新型的核苷酸结合基序。其他初步数据已经证实了酵母SRX作用的提议方案的某些方面，尽管我们的结果中有几个惊喜（隔离二硫键键入而不是硫代硫酸酯连接的中间体，并且需要进一步研究glutathione而不是胸膜蛋白的SRX：PRX复合物）。 该提案的目标是：确定与辅助因子和人类PRX复合物中人类SRX的晶体结构（AIM 1）；并进行稳态和部分周转实验以及位置定向的诱变，以阐明中间体的性质和反应速率，并表征对催化至关重要的残基（AIM 2＆3）。这些研究将对我们对硫酸还原酶作用的分子起源以及参与此过程的新型硫化学作用的理解产生重大贡献。