Intermediates during the fatty acyl CoA dehydrogenase catalyzed reduction of electron transfer flavoprotein (ETF) by fatty acyl CoA esters.
Intermediates during the fatty acyl CoA dehydrogenase catalyzed reduction of electron transfer flavoprotein (ETF) by fatty acyl CoA esters.
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脂肪酰辅酶 A 脱氢酶催化脂肪酰辅酶 A 酯还原电子转移黄素蛋白 (ETF) 过程中的中间体。
DOI：
10.1016/0006-291x(80)90576-8
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发表时间：
1980
影响因子：
3.1
通讯作者：
J. McFarland
中科院分区：
文献类型：
--
作者：
J. Reinsch;B. Feinberg;J. McFarland
传统上，通过利用电子从ETF的还原FAD转移到2，6-二氯靛酚（DCI）的染料测定来研究由饱和脂肪酰基CoA酯还原ETF的反应动力学。我们已经发现，它是最好的使用自然的荧光和吸收特性的ETF本身，以调查这两个稳态和单周转减少的ETF。我们的研究表明，形成一个红色的阴离子半醌作为中间体在两个电子还原的ETF在稳态动力学条件下使用丁酰CoA作为底物。此外，从370 nm吸光度增加或510 nm处ETF荧光发射减少（脱氢酶催化剂不发荧光）观察到的该半醌形成的稳态动力学数据计算的Vmax E =-6.0 sec-1比氢醌形成的Vmax E =-1.0 sec-1快得多。尽管DCI是一个双电子受体，但丁酰CoA的Vmax E =-4.0 sec− 1受到ETF半醌形成速率的限制，因此ETF半醌必须将其单电子转移到DCI。通过将电子定量地转移到脱氢酶催化剂的底物对ETF还原成半醌的程度（从荧光测量）的研究表明，总体化学计量是两个电子转移到二聚ETF分子上的两个FAD黄素。此外，对苯二酚不是由ETF半醌二聚体的反硝化形成的，因为对苯二酚的形成并不伴随着从这种反硝化预测的荧光增加。
The reaction kinetics for the reduction of ETF by saturated fatty acyl CoA esters has traditionally been studied by dye assays utilizing transfer of electrons from the reduced FAD of ETF to 2, 6-Dichloroindophenol (DCI). We have found that it is preferable to use the natural fluorescence and absorbance properties of ETF itself in order to investigate both the steady state and single turnover reduction of ETF. Our investigations indicate formation of a red anionic semiquinone as an intermediate in the two electron reduction of ETF under steady state kinetic condition using butyryl CoA as substrate. Furthermore, V max E∼-6.0 sec− 1 calculated from steady state kinetic data for formation of this semiquinone observed from the increase in 370 nm absorbance or decrease in ETF fluorescence emission at 510 nm (the dehydrogenase catalyst does not fluoresce) is much faster than V max E∼-1.0 sec− 1 for hydroquinone formation. Even though DCI is a two electron acceptor, V max E∼-4.0 sec− 1 for butyryl CoA is limited by the rate of formation of ETF semiquinone, which must, therefore, transfer its single electron to DCI. Investigation of the extent of ETF reduction to semiquinone (measured from fluorescence) by a substrate which transfers electrons quantitatively to the dehydrogenase catalyst indicates that the overall stoichiometry is two electrons transferred to the two FAD flavins on the dimeric ETF molecule. Furthermore, hydroquinone is not formed from disproportionation of the ETF semiquinone dimer since formation of hydroquinone is not accompanied by the fluorescence increase predicted from such disproportionation.