Westover J B, Goodman S I, Frerman F E
The Program in Human Medical Genetics, The Department of Pediatrics, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
Biochemistry. 2001 Nov 20;40(46):14106-14. doi: 10.1021/bi015637p.
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 a k(cat) of 2.7 s(-1). The k(cat) in the overall oxidation-decarboxylation reaction at pH 7.6 is about 9 s(-1). The binding of glutaconyl-CoA was quantitatively assessed from the K(m) in the hydratase reaction, 3 microM, and the K(i), 1.0 microM, as a competitive inhibitor of the dehydrogenase. These values compare with K(m) and K(i) of 4.0 and 12.9 microM, respectively, for crotonyl-CoA. Glu370 is the general base catalyst in the dehydrogenase that abstracts an alpha-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 (lambda(max) approximately 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.
戊二酰辅酶A（CoA）被认为是戊二酰辅酶A脱氢酶催化的戊二酰辅酶A氧化脱羧反应中与酶结合的中间体。我们在用戊二酰辅酶A对脱氢酶进行厌氧还原后，证明了戊二酰辅酶A与戊二酰辅酶A脱氢酶结合。戊二酰辅酶A脱氢酶还具有内在的烯酰辅酶A水合酶活性，这是酰基辅酶A脱氢酶家族其他成员的特性。该酶在pH 7.6时能迅速使戊二酰辅酶A水合，催化常数（k(cat)）为2.7 s(-1)。在pH 7.6的总体氧化脱羧反应中，k(cat)约为9 s(-1)。从水合酶反应中的米氏常数（K(m)）为3 microM以及作为脱氢酶竞争性抑制剂的抑制常数（K(i)）为1.0 microM定量评估了戊二酰辅酶A的结合情况。这些值与巴豆酰辅酶A的K(m)和K(i)分别为4.0和12.9 microM相比。Glu370是脱氢酶中的通用碱催化剂，它提取底物的α-质子以启动催化途径。突变型脱氢酶Glu370Gln在脱氢和水合酶反应中无活性。然而，这种突变型脱氢酶能使戊二酰辅酶A脱羧生成巴豆酰辅酶A，而无需脱氢酶黄素的氧化还原反应。向这种突变型脱氢酶中添加戊二酰辅酶A会导致长波长吸光度（λ(max)约为725 nm）迅速、短暂增加，并且发现巴豆酰辅酶A是唯一产物。我们提出这种吸收725 nm光的物质是脱羧后离域的巴豆酰辅酶A阴离子，其衰减是由于在没有通用酸催化剂Glu370(H(+))的情况下阴离子缓慢质子化的结果。在没有可检测到的氧化还原反应的情况下，数据表明脱氢酶黄素的氧化还原对于戊二酰辅酶A的脱羧不是必需
