Dakoji S, Shin I, Battaile K P, Vockley J, Liu H W
Department of Chemistry, University of Minnesota, Minneapolis 55455, USA.
Bioorg Med Chem. 1997 Dec;5(12):2157-64. doi: 10.1016/s0968-0896(97)00159-4.
The acyl-CoA dehydrogenases are a family of related enzymes that share high structural homology and a common catalytic mechanism which involves abstraction of an alpha-proton from the substrate by an active site glutamate residue. Several lines of investigation have shown that the position of the catalytic glutamate is conserved in most of these dehydrogenases (the E2 site), but is in a different location in two other family members (the E1 site). Using site specific in vitro mutagenesis, a double mutant rat short chain acyl-CoA dehydrogenase (rSCAD) has been constructed in which the catalytic glutamate is moved from the E2 to the E1 site (Glu368Gly/Gly247Glu). This mutant enzyme is catalytically active, but utilizes substrate less efficiently than the native enzyme (K(m) = 0.6 and 2.0 microM, and Vmax = 2.8 and 0.3 s-1 for native and mutant enzyme respectively). In this study we show that both the wild-type and mutant rSCADs display identical stereochemical preference for catalysis--abstraction of the alpha-HR from the substrate followed by transfer of the beta-HR to the FAD coenzyme. These results, in conjunction with molecular modeling of the native and double mutant SCAD indicate that the catalytic base in the E1 and E2 sites are topologically similar and catalytically competent. However, analysis of the 1H NMR spectra of the incubation products of these two enzymes revealed that, in contrast to the wild-type rSCAD, the Gly368Glu/Gly247Glu rSCAD could not perform gamma-proton exchange of the product with the solvent, a property inherent to most acyl-CoA dehydrogenases. It is evident that the base in the mutant enzyme has access to the alpha-HR but is far removed from the gamma-Hs. These findings provide further support for a one base mechanism of alpha- and gamma-reprotonation/deprotonation catalysis by acyl-CoA dehydrogenases.
酰基辅酶A脱氢酶是一类相关酶，它们具有高度的结构同源性和共同的催化机制，该机制涉及活性位点的谷氨酸残基从底物中提取α-质子。多项研究表明，大多数这些脱氢酶中催化谷氨酸的位置是保守的（E2位点），但在另外两个家族成员中位于不同位置（E1位点）。利用位点特异性体外诱变技术，构建了一种双突变大鼠短链酰基辅酶A脱氢酶（rSCAD），其中催化谷氨酸从E2位点移至E1位点（Glu368Gly/Gly247Glu）。这种突变酶具有催化活性，但与天然酶相比，利用底物的效率较低（天然酶和突变酶的Km分别为0.6和2.0微摩尔，Vmax分别为2.8和0.3秒-1）。在本研究中，我们表明野生型和突变型rSCAD在催化方面表现出相同的立体化学偏好——从底物中提取α-HR，然后将β-HR转移到FAD辅酶上。这些结果，结合天然和双突变SCAD的分子模型表明，E1和E2位点的催化碱基在拓扑结构上相似且具有催化活性。然而，对这两种酶的孵育产物的1H NMR光谱分析表明，与野生型rSCAD不同，Gly368Glu/Gly247Glu rSCAD不能使产物与溶剂进行γ-质子交换，这是大多数酰基辅酶A脱氢酶固有的特性。显然，突变酶中的碱基可以接触到α-HR，但与γ-Hs相距甚远。这些发现为酰基辅酶A脱氢酶催化α-和γ-再质子化/去质子化的单碱基机制提供了进一步的支持。
