Ramaswamy S, Eklund H, Plapp B V
Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala.
Biochemistry. 1994 May 3;33(17):5230-7. doi: 10.1021/bi00183a028.
Structures of the enzyme complexed with NAD+ and 2,3,4,5,6-pentafluorobenzyl alcohol were determined by X-ray crystallography at a resolution of 2.1 A and to a refinement R value of 18.3% for a monoclinic (P2(1)) form and to 2.4 A and an R value of 18.9% for a triclinic crystal form. The pentafluorobenzyl alcohol does not react, due to electron withdrawal by the fluorine atoms. A structure with NAD+ and p-bromobenzyl alcohol in the monoclinic form was also determined at 2.5 A and an R value of 16.7%. The conformations of the subunits in the monoclinic and triclinic crystal forms are very similar. The dimer is the asymmetric unit, and a rigid body rotation closes the cleft between the coenzyme and catalytic domains upon complex formation. In the monoclinic form, this conformational change is described by a rotation of 9 degrees in one subunit and 10 degrees in the other. The pentafluoro- and p-bromobenzyl alcohols bind in overlapping positions. The hydroxyl group of each alcohol is ligated to the catalytic zinc and participates in an extensive hydrogen-bonded network that includes the imidazole group of His-51, which can act as a base and shuttle a proton to solvent. The hydroxymethyl carbon of the pentafluorobenzyl alcohol is 3.4 A from C4 of the nicotinamide ring, and the pro-R hydrogen is in a good position for direct transfer to C4. The p-bromobenzyl alcohol may react after small rotations around single bonds of the alcohol. These structures should approximate the active Michaelis-Menten complexes.
通过X射线晶体学确定了与NAD⁺和2,3,4,5,6 - 五氟苄醇复合的酶的结构，单斜晶系（P2(1)）形式的分辨率为2.1 Å，精修R值为18.3%，三斜晶系晶体形式的分辨率为2.4 Å，R值为18.9%。由于氟原子的吸电子作用，五氟苄醇不发生反应。还确定了单斜晶系形式中与NAD⁺和对溴苄醇的结构，分辨率为2.5 Å，R值为16.7%。单斜晶系和三斜晶系晶体形式中亚基的构象非常相似。二聚体是不对称单元，复合物形成时，刚体旋转会闭合辅酶和催化结构域之间的裂隙。在单斜晶系形式中，这种构象变化表现为一个亚基旋转9度，另一个亚基旋转10度。五氟苄醇和对溴苄醇在重叠位置结合。每种醇的羟基与催化锌相连，并参与一个广泛的氢键网络，该网络包括His - 51的咪唑基团，其可作为碱并将质子传递给溶剂。五氟苄醇的羟甲基碳距离烟酰胺环的C4为3.4 Å，前R氢处于直接转移至C4的良好位置。对溴苄醇可能在围绕醇的单键进行小角度旋转后发生反应。这些结构应近似于活性米氏复合物。
