Rubinson J F, Corbin J L, Burgess B K
Biochemistry. 1983 Dec 20;22(26):6260-8. doi: 10.1021/bi00295a034.
We have examined the interaction of methyl isocyanide with the purified component proteins of Azotobacter vinelandii nitrogenase (Av1 and Av2). CH3NC was shown to be a potent reversible inhibitor (Ki = 158 microM) of total electron flow, apparently uncoupling magnesium adenosine 5'-triphosphate hydrolysis from electron transfer to substrate. CH3NC is a substrate (Km = 0.688 mM at Av2/Av1 = 8), and extrapolation of the data indicates that at high enough CH3NC concentration, H2 evolution can be eliminated. The products are methane plus methylamine (six electrons) and dimethylamine (four electrons). There is an excess (relative to methane) of methylamine formed, which may arise by hydrolysis of a two-electron intermediate. A rapid high-performance liquid chromatography/fluorescence method was developed for methylamine determination. The products C2H4 and C2H6 appear to be formed via a reduction followed by an insertion mechanism. CH3NC appears to be reduced at an enzyme state more oxidized than the one responsible for H2 evolution or N2 reduction. Other substrates (C2H2 greater than N2 congruent to azide greater than N2O) all both relieve CH3NC inhibition and inhibit CH3NC reduction. Both effects occur in the same relative order, implying productive (substrate) and nonproductive (inhibitor) modes of binding of CH3NC to the same site.
我们研究了甲基异氰化物与棕色固氮菌固氮酶（Av1和Av2）的纯化组分蛋白之间的相互作用。结果表明，CH3NC是总电子流的一种强效可逆抑制剂（Ki = 158 microM），显然能使镁腺苷5'-三磷酸水解与向底物的电子转移解偶联。CH3NC是一种底物（在Av2/Av1 = 8时Km = 0.688 mM），数据外推表明，在足够高的CH3NC浓度下，氢气释放可被消除。产物是甲烷加甲胺（六个电子）和二甲胺（四个电子）。形成的甲胺相对于甲烷有过量，这可能是由双电子中间体水解产生的。开发了一种快速高效液相色谱/荧光法用于甲胺测定。产物C2H4和C2H6似乎是通过还原后插入机制形成的。CH3NC似乎在比负责氢气释放或氮气还原的酶状态更氧化的状态下被还原。其他底物（C2H2大于N2等同于叠氮化物大于N2O）都既能减轻CH3NC抑制又能抑制CH3NC还原。两种效应以相同的相对顺序发生，这意味着CH3NC以生产性（底物）和非生产性（抑制剂）模式结合到同一位点。
