Possible radical mechanism in enzymatic carboxylation of phenol

苯酚酶促羧化的可能自由基机制

• 批准号: 5313758
• 负责人: Professor Dr. Georg Fuchs
• 金额: --
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2001
• 资助国家: 德国
• 起止时间: 2000-12-31 至 2005-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5313758?language=en
• 关键词: Possible; radical; mechanism; enzymatic; carboxylation

Anaerobic metabolism of phenol proceeds via para carboxylation to 4-hydroxybenzoate. This endergonic reaction is known since long in chemistry (Kolbe-Schmitt-carboxylation), but is only poorly studied in biology. The phenol carboxylating enzyme system in the bacterium Thauera aromatica will be investigated. It does not belong to any group of the studied carboxylases as it proceeds in two steps via a phosphorylated free intermediate (phenylphosphate) and uses a metal (Mn2+) as co-catalyst and carbon dioxide as substrate. While phosphorylation of phenol seems essential to drive carboxylation forward, it makes phenylphosphate a poor substrate for CO2 attack. The phenol carboxylating system does not depend on biotin or thiamine diphosphate and is complex; 10 proteins are phenol-induced. The carboxylase belongs to a family of proteins whose functions are largely unknown. Most remarkably, the enzyme is extremely sensitive to oxygen and radical trapping agents. The phenyl-phosphate carboxylating enzyme will be purified and characterized. Special focus will be on the catalytic mechanism which according to our working hypothesis involves a strongly enzyme-bound phenolate intermediate and a radical species.

苯酚的厌氧代谢通过帕拉羧化反应生成4-羟基苯甲酸酯。这种吸能反应在化学上早已为人所知（Kolbe-Schmitt-carboxylation），但在生物学上研究甚少。本文研究了Thauera aromatica细菌中的酚羧化酶系统。它不属于任何一组研究的羧化酶，因为它在两个步骤中通过磷酸化的游离中间体（磷酸苯酯）进行，并使用金属（Mn 2+）作为助催化剂和二氧化碳作为底物。虽然苯酚的磷酸化似乎对推动羧化至关重要，但它使磷酸苯酯成为CO2攻击的不良底物。苯酚羧化系统不依赖于生物素或硫胺素二磷酸，是复杂的; 10种蛋白质是苯酚诱导的。羧化酶属于一个蛋白质家族，其功能在很大程度上是未知的。最值得注意的是，这种酶对氧和自由基捕获剂极其敏感。将对磷酸苯酯羧化酶进行纯化和表征。特别关注的是催化机制，根据我们的工作假设，涉及一个强烈的酶结合的酚盐中间体和自由基物种。