Illarionov B, Eisenreich W, Bacher A
Lehrstuhl für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstrasse 4, D-85747 Garching, Germany.
Proc Natl Acad Sci U S A. 2001 Jun 19;98(13):7224-9. doi: 10.1073/pnas.131610698. Epub 2001 Jun 12.
The S41A mutant of riboflavin synthase from Escherichia coli catalyzes the formation of riboflavin from 6,7-dimethyl-8-ribityllumazine at a very low rate. Quenching of presteady-state reaction mixtures with trifluoroacetic acid afforded a compound with an absorption maximum at 412 nm (pH 1.0) that can be converted to a mixture of riboflavin and 6,7-dimethyl-8-ribityllumazine by treatment with wild-type riboflavin synthase. The compound was shown to qualify as a kinetically competent intermediate of the riboflavin synthase-catalyzed reaction. Multinuclear NMR spectroscopy, using various 13C- and 15N-labeled samples, revealed a pentacyclic structure arising by dimerization of 6,7-dimethyl-8-ribityllumazine. Enzyme-catalyzed fragmentation of this compound under formation of riboflavin can occur easily by a sequence of two elimination reactions.
来自大肠杆菌的核黄素合酶的S41A突变体以非常低的速率催化由6,7-二甲基-8-核糖基卢马嗪形成核黄素。用三氟乙酸淬灭预稳态反应混合物得到一种在412nm(pH 1.0)处具有最大吸收的化合物,该化合物通过用野生型核黄素合酶处理可转化为核黄素和6,7-二甲基-8-核糖基卢马嗪的混合物。该化合物被证明是核黄素合酶催化反应的动力学活性中间体。使用各种13C和15N标记的样品进行的多核NMR光谱显示,由6,7-二甲基-8-核糖基卢马嗪二聚化产生的五环结构。在形成核黄素的过程中,该化合物的酶催化断裂可以通过一系列两个消除反应轻松发生。