Radical reactions in fermentation pathways of nitrogenous compounds

含氮化合物发酵途径中的自由基反应

• 批准号: 5178032
• 负责人: Professor Dr. Wolfgang Buckel
• 金额: --
• 依托单位: School of Chemistry (aufgelöst)
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 1999
• 资助国家: 德国
• 起止时间: 1998-12-31 至 2006-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5178032?language=en
• 关键词: Radical; reactions; fermentation; pathways; nitrogenous

The mechanisms of the coenzyme B12-dependent glutamate and 2-methyleneglutarate mutase, as well as the [4Fe-4S]cluster- and FAD-dependent 4-hydroxybutyryl-CoA dehydratase, will be investigated. It is proposed that catalysis by all three clostridial enzymes is a consequence if hydrogen abstraction. Kinetic isotope effects and newly designed inhibitors will be used to determine the rate limiting steps. The use of such inhibitors may cause the radical intermediates to persist in sufficient concentrations and enable their analysis by EPR and ENDOR spectrocsopy. Therefore, a series of substrates, have to be synthesised. The formation of a methyl group by each enzyme will analysed by the chiral acetate methodology. Determination of the steric course of the b,g-elimination of water from 4-hydroxybutyryl-CoA to crotonyl-CoA may tell whether the reaction occurs in a stepwise or concerted manner. Mutagenesis of essential amino acid residues, identified by the emerging three-dimenional structures, should help to define the role of the proteins in these catalyses.

将研究辅酶B12依赖性谷氨酸和2-亚甲基戊二酸脱氢酶以及[4Fe-4S]簇和FAD依赖性4-羟基丁酰辅酶A脱氢酶的机制。有人提出，催化的所有三个梭菌酶是一个后果，如果氢提取。动力学同位素效应和新设计的抑制剂将用于确定限速步骤。使用这种抑制剂可能会导致自由基中间体在足够的浓度下持续存在，并能够通过EPR和ENDOR光谱进行分析。因此，必须合成一系列底物。通过手性乙酸酯方法分析每种酶形成甲基的情况。测定水从4-羟基丁酰-CoA到巴豆酰-CoA的B，g-消除的空间过程可以判断反应是以逐步方式还是协同方式发生。必需氨基酸残基的突变，通过新兴的三维结构鉴定，应该有助于确定蛋白质在这些催化中的作用。