Binding, hydration, and decarboxylation of the reaction intermediate glutaconyl-coenzyme A by human glutaryl-CoA dehydrogenase.
Binding, hydration, and decarboxylation of the reaction intermediate glutaconyl-coenzyme A by human glutaryl-CoA dehydrogenase.
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人戊二酰辅酶 A 脱氢酶对反应中间体戊二酰辅酶 A 的结合、水合和脱羧。
DOI：
10.1021/bi015637p
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发表时间：
2001
期刊：
影响因子：
2.9
通讯作者：
Frerman,FE
中科院分区：
文献类型：
--
作者：
Westover,JB;Goodman,SI;Frerman,FE
戊烯二酰辅酶A（CoA）是戊二酰辅酶A脱氢酶催化的戊二酰辅酶A氧化脱羧反应中的假定酶结合中间体。我们证明了戊烯二酰辅酶A结合戊二酰辅酶A脱氢酶后，厌氧还原的脱氢酶与戊二酰辅酶A。戊二酰辅酶A脱氢酶还具有固有的烯酰辅酶A水合酶活性，这是酰基辅酶A脱氢酶家族的其他成员的特性。该酶在pH 7.6下以2.7 s-1的akcatof快速水合戊烯二酰辅酶A。在pH 7.6时，总氧化-脱羧反应的kcatin约为9 s-1。根据水合酶反应的Kmin（3 μM）和作为脱氢酶竞争性抑制剂的Ki（1.0 μM）定量评估戊烯二酰辅酶A的结合。这些值与巴豆酰辅酶A的KmandKi分别为4.0和12.9 μM相比。Glu 370是脱氢酶中的一般碱催化剂，其提取底物的α-质子以启动催化途径。突变型脱氢酶Glu 370 Gln在脱氢酶和水合酶反应中无活性。然而，这种突变的脱氢酶将戊烯二酰辅酶A脱羧为巴豆酰辅酶A，而没有脱氢酶黄素的氧化还原反应。将戊烯二酰辅酶A添加到该突变脱氢酶中导致长波长吸光度（λmax = 725 nm）快速、短暂增加，并且发现巴豆酰辅酶A为唯一产物。我们提出，这725 nm的吸收物种是离域的巴豆酰辅酶A阴离子，脱羧和衰减是在一般的酸催化剂，Glu 370（H+）的情况下的阴离子的缓慢质子化的结果。在没有可检测的氧化-还原的情况下，数据表明脱氢酶黄素的氧化-还原对于戊烯二酰辅酶A的脱羧不是必需的。
Glutaconyl-coenzyme A (CoA) is the presumed enzyme-bound intermediate in the oxidative decarboxylation of glutaryl-CoA that is catalyzed by glutaryl-CoA dehydrogenase. We demonstrated glutaconyl-CoA bound to glutaryl-CoA dehydrogenase after anaerobic reduction of the dehydrogenase with glutaryl-CoA. Glutaryl-CoA dehydrogenase also has intrinsic enoyl-CoA hydratase activity, a property of other members of the acyl-CoA dehydrogenase family. The enzyme rapidly hydrates glutaconyl-CoA at pH 7.6 with akcatof 2.7 s-1. Thekcatin the overall oxidation−decarboxylation reaction at pH 7.6 is about 9 s-1. The binding of glutaconyl-CoA was quantitatively assessed from theKmin the hydratase reaction, 3 μM, and theKi, 1.0 μM, as a competitive inhibitor of the dehydrogenase. These values compare withKmandKiof 4.0 and 12.9 μM, respectively, for crotonyl-CoA. Glu370 is the general base catalyst in the dehydrogenase that abstracts an α-proton of the substrate to initiate the catalytic pathway. The mutant dehydrogenase, Glu370Gln, is inactive in the dehydrogenation and the hydratase reactions. However, this mutant dehydrogenase decarboxylates glutaconyl-CoA to crotonyl-CoA without oxidation−reduction reactions of the dehydrogenase flavin. Addition of glutaconyl-CoA to this mutant dehydrogenase results in a rapid, transient increase in long-wavelength absorbance (λmax≈ 725 nm), and crotonyl-CoA is found as the sole product. We propose that this 725 nm-absorbing species is the delocalized crotonyl-CoA anion that follows decarboxylation and that the decay is the result of slow protonation of the anion in the absence of the general acid catalyst, Glu370(H+). In the absence of detectable oxidation−reduction, the data indicate that oxidation−reduction of the dehydrogenase flavin is not essential for decarboxylation of glutaconyl-CoA.