Kis K, Bacher A
Department of Organic Chemistry and Biochemistry, Technical University of Munich, Garching, Federal Republic of Germany.
J Biol Chem. 1995 Jul 14;270(28):16788-95. doi: 10.1074/jbc.270.28.16788.
The lumazine synthase/riboflavin synthase complex of Bacillus subtilis consists of an icosahedral capsid of 60 beta subunits surrounding a core of three alpha subunits. The beta subunits catalyze the condensation of 5-amino-6-ribityl-amino-2,4(1H,3H)-pyrimidinedione (PYR) with 3,4-dihydroxy-2-butanone 4-phosphate (DHB) yielding 6,7-dimethyl-8-ribityllumazine. This intermediate is converted to riboflavin by the alpha subunits via an unusual dismutation. The second product of this reaction is PYR, which is also a substrate of the beta subunits and can be recycled in the catalytic process. Sigmoidal kinetics would be expected for the formation of riboflavin from PYR and DHB and are indeed observed with mixtures of artifactual beta 60 capsids and alpha subunit trimers. In contrast, the formation of riboflavin from PYR and DHB by the native alpha 3 beta 60 is characterized by a finite initial rate, which is similar to the rate of lumazine formation. Most notably, the rate of riboflavin formation has its maximum value at t = 0 and decreases dramatically after the consumption of PYR and DHB despite the presence of transiently formed lumazine. These data suggest that a significant fraction of DHB is converted to riboflavin by substrate channeling, which is conducive to an improved overall catalytic rate of riboflavin formation at low substrate concentrations. The channel is leaky, and the intermediate lumazine is therefore transiently accumulated in the bulk solution. The partitioning factor relating the direct formation of riboflavin via substrate channeling and the formation of transient 6,7-dimethyl-8-ribityl-lumazine increases at low concentrations of the substrates PYR and DHB and has a maximum value at pH 7.5. Channeling appears to result from the compartmentalization of the alpha subunits inside the icosahedral beta subunit capsid whose catalytic sites are located close to the inner capsid surface.
枯草芽孢杆菌的鲁玛嗪合酶/核黄素合酶复合物由一个由60个β亚基组成的二十面体衣壳围绕三个α亚基的核心构成。β亚基催化5-氨基-6-核糖基氨基-2,4(1H,3H)-嘧啶二酮（PYR）与3,4-二羟基-2-丁酮4-磷酸（DHB）缩合生成6,7-二甲基-8-核糖基鲁玛嗪。该中间体通过α亚基经一种不寻常的歧化反应转化为核黄素。该反应的第二个产物是PYR，它也是β亚基的底物，可在催化过程中循环利用。预计从PYR和DHB形成核黄素会呈现S形动力学，并且在人工β60衣壳和α亚基三聚体的混合物中确实观察到了这种情况。相比之下，天然α3β60从PYR和DHB形成核黄素的特征是具有有限的初始速率，这与鲁玛嗪形成的速率相似。最值得注意的是，核黄素形成的速率在t = 0时达到最大值，并且在PYR和DHB消耗后尽管存在瞬时形成的鲁玛嗪，但仍会急剧下降。这些数据表明，相当一部分DHB通过底物通道化转化为核黄素，这有利于在低底物浓度下提高核黄素形成的总体催化速率。该通道是有泄漏的，因此中间体鲁玛嗪会在本体溶液中瞬时积累。在底物PYR和DHB浓度较低时，通过底物通道化直接形成核黄素与瞬时形成6,7-二甲基-8-核糖基鲁玛嗪的分配系数会增加，并且在pH 7.5时达到最大值。通道化似乎是由于二十面体β亚基衣壳内部α亚基的区室化导致的，其催化位点位于衣壳内表面附近。
