Participation of the iron-sulphur cluster and of the covalently bound coenzyme of trimethylamine dehydrogenase in catalysis.
Participation of the iron-sulphur cluster and of the covalently bound coenzyme of trimethylamine dehydrogenase in catalysis.
复制标题
铁硫簇和三甲胺脱氢酶的共价结合辅酶参与催化。
作者：
D. J. Steenkamp;T. Singer
细菌三甲胺脱氢酶含有新型共价结合的黄素单核苷酸和四聚体铁硫中心。脱氢酶吸收 1.5mol 连二亚硫酸盐/mol 酶，从而转化为黄素对苯二酚还原 (4Fe-4S) 形式，预期黄素的可见吸收带会漂白，并在电子顺磁共振谱中出现典型还原铁氧还蛋白的信号。然而，在用稍微过量的底物进行还原时，异常的吸收和电子顺磁共振光谱很快出现。后者归因于还原的 (4Fe-4S) 中心和黄素半醌之间的广泛相互作用。通过快速冷冻 e.p.r. 的组合研究了催化循环期间产生的酶的种类。和停流分光光度法。黄素最初还原为对苯二酚形式的速度太快，不足以限制催化速度，吩嗪硫酸甲酯对底物还原酶的再氧化也是如此。然而，从二氢黄素形成自旋-自旋相互作用物质的速度要慢得多，并且它可能是催化循环中的限速步骤，因为其形成速率与稳态动力学测定中确定的催化中心活性相当吻合。除了相互作用的形式外，在三甲胺还原过程中还发现了酶的第二种形式，其吸收光谱不同，其结构仍有待确定。
Bacterial trimethylamine dehydrogenase contains a novel type of covalently bound flavin mononucleotide and a tetrameric iron-sulphur centre. The dehydrogenase takes up 1.5mol of dithionite/mol of enzyme and is thereby converted into the flavin quinol-reduced (4Fe-4S) form, with the expected bleaching of the visible absorption band of the flavin and the emergence of signals of typical reduced ferredoxin in the electronparamagnetic-resonance spectrum. On reduction with a slight excess of substrate, however, unusual absorption and electron-paramagnetic-resonance spectra appear quite rapidly. The latter is attributed to extensive interaction between the reduced (4Fe-4S) centre and the flavin semiquinone. The species of enzyme arising during the catalytic cycle were studied by a combination of rapid-freeze e.p.r. and stopped-flow spectophotometry. The initial reduction of the flavin to the quinol form is far too rapid to be rate-limiting in catalysis, as is the reoxidation of the substrate-reduced enzyme by phenazine methosulphate. Formation of the spin-spin-interacting species from the dihydroflavin is considerably slower, however, and it may be the rate-limiting step in the catalytic cycle, since its rate of formation agrees reasonably well with the catalytic-centre activity determined in steady-state kinetic assays. In addition to the interacting form, a second form of the enzyme was noted during reduction by trimethylamine, differing in absorption spectrum, the structure of which remains to be determined.