REFINED STRUCTURE OF GLUTATHIONE-REDUCTASE AT 1.54 A RESOLUTION
REFINED STRUCTURE OF GLUTATHIONE-REDUCTASE AT 1.54 A RESOLUTION
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DOI：
10.1016/0022-2836(87)90191-4
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发表时间：
1987-06-05
影响因子：
5.6
通讯作者：
SCHULZ, GE
中科院分区：
文献类型：
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作者：
KARPLUS, PA;SCHULZ, GE
人谷胱甘肽还原酶的晶体结构已确定为 1.54 .ANG。使用约束最小二乘细化方法进行分辨率。基于 77,690 次优于 10 .ANG 的独立反射。分辨率，最终 R 因子为 18.6%，模型遵循 0.025 .ANG 内的标准几何形状。键长和2.4°。在键角中。最终的2Fo-Fc电子密度图允许在温度因子低于约25.ANG.2的情况下区分碳、氮和氧原子。除了 461 个氨基酸残基和辅基 FAD 之外，该模型还包含 524 个溶剂分子，其中约 118 个可以被认为是酶的组成部分。最大的溶剂簇位于二聚体界面处，包含 104 个互连的溶剂分子，其中部分分子组织成翘曲的片状结构。主链二面角集中在拉马钱德兰图的允许区域中。 β-折叠片中二面角的分布远大于α-螺旋，特别是α-螺旋核，表明β-结构具有更高的可塑性。分析显示存在大量的310-螺旋。侧链构象聚集在交错的位置，并显示出明确的偏好。此外，还观察到侧链的迁移率梯度。非极性和极性侧链显示每个键的平均温度因子分别增加 10% 和 25%。已检测到内部侧链的许多替代构象，特别是丝氨酸和甲硫氨酸。延伸的 FAD 分子还显示出非常刚性的黄素 ((B) = 8.7.ANG.2) 和更具流动性的腺嘌呤 ((B) = 16.2.ANG.2) 之间的迁移率梯度。整个活动中心秩序特别良好，温度系数约为 10 ANG.2。二聚体界面由刚性接触区域和柔性区域组成，刚性接触区域在大肠杆菌酶中得到很好的保守，而柔性区域则不然。总而言之，晶体接触处的埋入表面是二聚体界面处的埋入表面的一半大，但特异性较低。精细的结构清楚地表明不存在补偿 FAD 焦磷酸部分电荷的埋藏阳离子。黄素与标准几何形状略有偏差，这可能是由多肽环境引起的。与早期的解释相反，黄素的原子 N5 可以容纳一个质子，并且可以想象该质子会转变为具有氧化还原活性的二硫化物。该二硫键的 Cys58 可以以类似于丝氨酸蛋白酶的“电荷中继系统”的方式被激活。
The crystal structure of human glutathione reductase has been established at 1.54 .ANG. resolution using a restrained least-squares refinement method. Based on 77,690 independent reflections of better than 10 .ANG. resolution, a final R-factor of 18.6% was obtained with a model obeying standard geometry within 0.025 .ANG. in bond lengths and 2.4.degree. in bond angles. The final 2Fo-Fc electron density map allows for the distinction of carbon, nitrogen and oxygen atoms with temperature factors below about 25 .ANG.2. Apart from 461 amino acid residues and the prosthetic group FAD, the model contains 524 solvent molecules, about 118 of which can be considered an integral part of the enzyme. The largest solvent cluster is at the dimer interface and contains 104 interconnected solvent molecules, part of which are organized in a warped sheet-like structure. The main-chain dihedral angles are well-concentrated in the allowed regions of the Ramachandran plot. The spread of dihedral angles in .beta.-pleated sheets is much larger than in .alpha.-helices and especially in .alpha.-helix cores, indicating the higher plasticity of .beta.-structures. The analysis revealed a large amount of 310-helix. The side-chain conformations cluster at the staggered positions, and show well-defined preferecnes. Also, a mobility gradient is observed for side-chains. Non-polar and polar side-chains show average temperature factor increases per bond of 10% and 25%, respectively. A number of alternative conformations of internal side-chains, in particular serines and methionines, have been detected. The extended FAD molecule also shows a mobility gradient between the very rigid flavin ((B) = 8.7 .ANG.2) and the more mobile adenine ((B) = 16.2 .ANG.2). The entire active center is particularly well ordered, with temperature factors around 10 .ANG.2. The dimer interface consists of a rigid contact area, which is well conserved in the Escherichia coli enzyme, and a flexible area that is not. Altogether, the buried surfaces at the crystal contacts are half as large as at the dimer interface, but less specific. The refined structure shows clearly that there are no buried cations compensating the charge of the pyrophosphate moiety of FAD. The flavin deviates slightly from standard geometry, which is possibly caused by the polypeptide environment. In contrast to an earlier interpretation, atom N5 of the flavin can accommodate a proton, and it is conceivable that this proton proceeds to the redox-active disulfide. Cys58 of this disulfide may be activated in manner similar to the ''''charge relay system'''' of serine proteases.