Mechanistic studies on the dehydrogenases of methylotrophic bacteria. 1. The influence of substrate binding to reduced trimethylamine dehydrogenase on the intramolecular electron transfer between its prosthetic groups.
Mechanistic studies on the dehydrogenases of methylotrophic bacteria. 1. The influence of substrate binding to reduced trimethylamine dehydrogenase on the intramolecular electron transfer between its prosthetic groups.
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甲基营养菌脱氢酶的机理研究。
DOI：
10.1042/bj2070233
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发表时间：
1982
期刊：
影响因子：
--
通讯作者：
Beinert,H
中科院分区：
文献类型：
--
作者：
Steenkamp,DJ;Beinert,H
细菌W3 A1的三甲胺脱氢酶被还原，形成三重态，其中来自底物的两个电子分布在[4Fe-4S]簇和6-S-半胱氨酰-FMN半醌之间。在pH 8.5的滴定实验中，每摩尔酶需要约1.0摩尔二甲胺或0.5摩尔三甲胺来滴定酶至终点。然而，在pH值小于8.0时，需要过量的三甲胺以获得g = 4 e.p.r.的最大产率。信号，三重态的特征，或在365 nm处的最大吸光度，其指示黄素半醌的形成。每摩尔酶的0.86摩尔的三甲基胺的结合可以通过凝胶色谱法检测。当在四甲基氯化铵存在下用连二亚硫酸盐滴定酶时，在吸收两个电子后达到终点，这引起三重态，而在四甲基氯化铵不存在下消耗三个电子以完全还原酶。该酶被四甲基氯化铵非竞争性抑制，双倒数图的斜率是抑制剂浓度的凹向上的函数。数据表明，存在的底物和其他胺的结合位点的减少酶，这提高了酶的比例在三重态。
The trimethylamine dehydrogenase of bacterium W3A1 is reduced with the formation of a triplet state in which two electrons, derived from the substrate, are distributed between the [4Fe-4S] cluster and 6-S-cysteinyl-FMN semiquinone. In titration experiments at pH 8.5 about 1.0 mol of dimethylamine or 0.5 mol of trimethylamine per mol of the enzyme is required to titrate the enzyme to an endpoint. At pH values less than 8.0, however, an excess of trimethylamine is required to obtain maximal yield of the g = 4 e.p.r. signal, characteristic of the triplet state, or maximal absorbance at 365 nm which indicates formation of the flavin semiquinone. The binding of 0.86 mol of trimethylamine per mol of the enzyme could be detected by a gel chromatographic method. When the enzyme is titrated with dithionite in the presence of tetramethylammonium chloride, an endpoint is reached after the uptake of two electrons which give rise to the triplet state, whereas three electrons are consumed in the absence of tetramethylammonium chloride to reduce the enzyme completely. The enzyme is inhibited noncompetitively by tetramethylammonium chloride and the slopes of double reciprocal plots are a concave upwards function of inhibitor concentration. The data indicate the presence of a binding site for the substrate and other amines on the reduced enzyme which enhances the proportion of enzyme in the triplet state.