Williamson G, Engel P C
Biochem J. 1984 Mar 1;218(2):521-9. doi: 10.1042/bj2180521.
The absorption coefficient of butyryl-CoA dehydrogenase from Megasphaera elsdenii at 450 nm is determined as 14.4 mM-1 X cm-1 in the CoA-free form and 14.2 mM-1 X cm-1 in the CoA-liganded form (both yellow). The latter value is considerably higher than the earlier published estimate. Phenazine ethosulphate offers great advantages over phenazine methosulphate as a coupling dye in the catalytic assay despite giving lower Vmax. values (506 min-1 as compared with 1250 min-1 under the conditions used). The phenazine ethosulphate assay is used to establish a pH optimum of 8.05 for oxidation of 100 microM-butyryl-CoA. The rates of oxidation of a range of straight-chain, branched-chain and alicyclic acyl thioesters are used to provide the following information. Only straight-chain acyl groups containing 4-6 carbon atoms are easily accommodated by the postulated hydrophobic pocket of the enzyme. C-3-substituted acyl-CoA thioesters are not oxidized at a significant rate, suggesting that the C-3 pro-S-hydrogen atom of straight-chain substrates is partially exposed to the solvent. Acyl-CoA thioesters with substitutions at C-2 are oxidized, though at a lower rate than their straight-chain counterparts. This implies that the C-2 pro-S-hydrogen atom of straight-chain substrates is partially exposed to the solvent. Saturated alicyclic carboxylic acyl-CoA thioesters with 4-7 carbon atoms in the ring are oxidized, with maximal activity for the cyclohexane derivative. This implies that optimal oxidation requires a true trans orientation of the two departing hydrogen atoms. The strain imposed by bound unsaturated alicyclic acyl thioesters strikingly perturbs the flavin visible-absorption spectrum, with the exception of the cyclohex-2-ene derivative, which forms a complex with similar spectral properties to those of the crotonyl-CoA complex. In the thiol moiety of thioester substrates the amide bond of N-acetylcysteamine is essential for both binding and catalysis. The adenosine structure contributes substantially to strong binding, but is less important in determining the catalytic rate.
埃氏巨球形菌中丁酰辅酶A脱氢酶在450 nm处的吸收系数，在无辅酶A形式下测定为14.4 mM⁻¹·cm⁻¹，在与辅酶A结合的形式下（均为黄色）为14.2 mM⁻¹·cm⁻¹。后一个值明显高于先前发表的估计值。在催化测定中，作为偶联染料，硫酸吩嗪比硫酸甲酯吩嗪具有很大优势，尽管其Vmax值较低（在所使用的条件下，分别为506 min⁻¹和1250 min⁻¹）。硫酸吩嗪测定法用于确定100 microM丁酰辅酶A氧化的最适pH为8.05。一系列直链、支链和脂环族酰基硫酯的氧化速率用于提供以下信息。只有含4至6个碳原子的直链酰基容易被该酶假定的疏水口袋容纳。C-3位取代的酰基辅酶A硫酯不以显著速率被氧化，这表明直链底物的C-3 pro-S-氢原子部分暴露于溶剂中。C-2位有取代的酰基辅酶A硫酯会被氧化，但其速率低于相应的直链硫酯。这意味着直链底物的C-2 pro-S-氢原子部分暴露于溶剂中。环中含4至7个碳原子的饱和脂环族羧酸酰基辅酶A硫酯会被氧化，环己烷衍生物的活性最高。这意味着最佳氧化需要两个离去氢原子的真正反式取向。结合的不饱和脂环族酰基硫酯所施加的张力显著干扰了黄素可见吸收光谱，环己-2-烯衍生物除外，它形成的复合物具有与巴豆酰辅酶A复合物相似的光谱特性。在硫酯底物的硫醇部分，N-乙酰半胱胺的酰胺键对结合和催化都至关重要。腺苷结构对强结合有很大贡献，但在决定催化速率方面不太重要。
