MECHANISTIC STUDIES WITH GENERAL ACYL-COA DEHYDROGENASE AND BUTYRYL-COA DEHYDROGENASE - EVIDENCE FOR THE TRANSFER OF THE BETA-HYDROGEN TO THE FLAVIN N(5)-POSITION AS A HYDRIDE
MECHANISTIC STUDIES WITH GENERAL ACYL-COA DEHYDROGENASE AND BUTYRYL-COA DEHYDROGENASE - EVIDENCE FOR THE TRANSFER OF THE BETA-HYDROGEN TO THE FLAVIN N(5)-POSITION AS A HYDRIDE
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DOI：
10.1021/bi00309a008
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发表时间：
1984-01-01
期刊：
影响因子：
2.9
通讯作者：
MASSEY, V
中科院分区：
文献类型：
--
作者：
GHISLA, S;THORPE, C;MASSEY, V
来自埃氏巨球酵母的丁酰辅酶A脱氢酶催化α-CoA的交换，和β-用溶剂除去底物中的氢。该交换的化学计量通过使用3 H2O标记确定为1.94 ±。0.1每个底物分子。在厌氧条件下，3 H标记掺入底物的速率是双相的，表明α-和β-氢以相同的速率交换。在2 H2O中的交换导致一个2 H各自结合到α-和β-通过伴随的1H NMR实验测定并通过质谱分析确认，相反，对于来自猪肾的一般酰基-CoA脱氢酶，仅α-CoA脱氢酶的交换是不可逆的。发现了氢。氢是在底物脱氢过程中转移（可逆地）到黄素5-位的氢。这通过5- 3 H-和5- 2 H-还原的5-脱氮-FAD-通用酰基-CoA脱氢酶与巴豆酰-CoA反应来证明。还原的黄素类似物只有一个面能够将氢转移到底物。该反应的速率对于5-脱氮-FAD-酶为11.1 s-1，对于[5- 2 H]脱氮-FAD-酶为2.2 s-1，产生同位素效应5。这些值与天然还原酶与巴豆酰辅酶A反应的速率2.6 s-1相比。因此，2种还原酶（正常与5-脱氮-FAD-酶）以相似的速率反应，表明机制相似。结果被解释为催化序列的证据，其中α-氢被提取为质子，随后排出β-氢作为氢化物并直接转移到黄素位置N（5）。
Butyryl-CoA dehydrogenase from Megasphaera elsdenii catalyzes the exchange of the .alpha.- and .beta.-hydrogens of substrate with solvent. The stoichiometry of this exchange was determined by using 3H2O label as 1.94 .+-. 0.1 per substrate molecule. The rate of 3H label incorporation into substrate under anaerobic conditions is monophasic, indicating that both the .alpha.- and .beta.-hydrogens exchange at the same rate. The exchange in 2H2O leads to incorporation of one 2H each into the .alpha.- and .beta.-positions of butyryl-CoA, as determined by companion 1H NMR experiments and confirmed by mass spectroscopic analysis. In contrast, with general acyl-CoA dehydrogenase from pig kidney, only exchange of the .alpha.-hydrogen was found. The .beta.-hydrogen is the one that is transferred (reversibly) to the flavin 5-position during substrate dehydrogenation. This was demonstrated by reacting 5-3H- and 5-2H-reduced 5-deaza-FAD-general acyl-CoA dehydrogenase with crotonyl-CoA. Only one face of the reduced flavin analog is capable of transferring hydrogen to substrate. The rate of this reaction is 11.1 s-1 for 5-deaza-FAD-enzyme and 2.2 s-1 for [5-2H]deaza-FAD-enzyme, yielding an isotope effect of 5. These values compare with a rate of 2.6 s-1 for the reaction of native reduced enzyme with crotonyl-CoA. The 2 reduced enzymes (normal vs. 5-deaza-FAD-enzyme) thus react at similar rates, indicating a similar mechanism. The results are interpreted as evidence for a catalytic sequence in which the .alpha.-hydrogen is abstracted as a proton, followed by expulsion of the .beta.-hydrogen as a hydride and its direct transfer to the flavin position N(5).