Kumar N R, Srivastava D K
Biochemistry Department, North Dakota State University, Fargo 58105, USA.
Biochemistry. 1995 Jul 25;34(29):9434-43. doi: 10.1021/bi00029a019.
In a previous paper, we demonstrated that the reductive half-reaction of medium-chain fatty acyl-CoA dehydrogenase (MCAD), utilizing octanoyl-CoA as physiological substrate, generates two (kinetically distinct) forms of the reduced enzyme (MCAD-FADH2) - octenoyl-CoA charge-transfer complexes [Kumar, N.R., & Srivastava, D.K. (1994) Biochemistry 33, 8833-8841]. We present evidence that octenoyl-CoA dissociates from the second (most stable) charge-transfer complex (referred to as CT2) via two alternative ("facile" and "restricted") pathways. The dissociation of octenoyl-CoA via the facile pathway involves the reversal of the overall reductive half-reaction of the enzyme, generating MCAD-FAD - octanoyl-CoA as the Michaelis complex, followed by dissociation of the latter complex into MCAD-FAD + octanoyl-CoA. Hence, via this pathway, octenoyl-CoA is released from the enzyme site in the form of octanoyl-CoA. In contrast, the restricted pathway involves a direct (albeit slow) dissociation of octenoyl-CoA from CT2 to yield MCAD-FADH2 + octenoyl-CoA. The kinetic profile for the dissociation of octenoyl-CoA via the restricted pathway matches the rate of oxidation of the reduced flavin (within CT2) by O2. This suggests that the oxidase activity of the enzyme remains suppressed as long as the reduced enzyme predominates in the form of the charge-transfer complex(es). The oxidase activity of the enzyme emerges concomitantly with the conversion of CT2 to the MCAD-FADH2 - octenoyl-CoA Michaelis complex. The energetic basis for the dissociation of octenoyl-CoA via the facile and restricted pathways and the mechanism of suppression of the oxidase activity of the enzyme are discussed.
在之前的一篇论文中，我们证明了中链脂肪酰辅酶A脱氢酶（MCAD）以辛酰辅酶A作为生理底物时的还原半反应会产生两种（动力学上不同的）还原酶形式（MCAD-FADH2）——辛烯酰辅酶A电荷转移复合物[库马尔，N.R.，& 斯里瓦斯塔瓦，D.K.（1994年）《生物化学》33卷，8833 - 8841页]。我们提供的证据表明，辛烯酰辅酶A通过两种替代途径（“容易的”和“受限的”）从第二种（最稳定的）电荷转移复合物（称为CT2）上解离。通过容易途径的辛烯酰辅酶A解离涉及酶的整体还原半反应的逆转，生成MCAD-FAD - 辛酰辅酶A作为米氏复合物，随后后者复合物解离为MCAD-FAD + 辛酰辅酶A。因此，通过这条途径，辛烯酰辅酶A以辛酰辅酶A的形式从酶位点释放。相比之下，受限途径涉及辛烯酰辅酶A从CT2直接（尽管缓慢）解离，生成MCAD-FADH2 + 辛烯酰辅酶A。通过受限途径的辛烯酰辅酶A解离的动力学曲线与O2对还原黄素（在CT2内）的氧化速率相匹配。这表明只要还原酶以电荷转移复合物的形式占主导，酶的氧化酶活性就会受到抑制。酶的氧化酶活性伴随着CT2向MCAD-FADH2 - 辛烯酰辅酶A米氏复合物的转化而出现。本文讨论了通过容易和受限途径的辛烯酰辅酶A解离的能量基础以及酶氧化酶活性的抑制机制。
