The 3-D Structure and Reaction Mechanism in The Highly Organized supra-Molecule of Pyruvate Dehydrogenase Complex.

丙酮酸脱氢酶复合体是一种高度组织化的多酶系统，由丙酮酸脱氢酶(E1)、二氢硫辛酰乙酰转移酶(E2)和硫胺脱氢酶(E3)三种酶的多个拷贝组成，特异性地、高效地催化不可逆的一系列反应。根据生物体的不同，将络合物分为两种类型的中心核心结构，即具有不同对称性的E2。一个在革兰氏阴性细菌中具有432对称性，另一个在革兰氏阳性细菌和真核生物中具有532对称性。对后一种类型的酵母分离的E3组分的晶体结构进行了测定。发现有两种不同类型的晶体，它们的结晶习性各不相同。用分子置换方法计算了晶体结构。通过对电子密度图进行平均化和扁平化，改善了初始相。用XploR对Frodo构筑的分子模型的原子坐标进行了修正，约束条件为非晶学的2重对称性。虽然分子堆积不同，但分子结构相似。与谷胱甘肽还原酶的进一步比较表明，尽管在C-末端结构域观察到较大的原子偏离，但两个结构域之间的活性部位具有几乎相同的三维结构。这种相似性也在具有432对称性的不同生物的E3中观察到。从目前的调查来看，E3的三级结构在本质上是相同的，即使在两种类型之间的不同架构中也是如此。值得注意的是，这种强烈的结构保守性可能是该酶对功能的抑制。

The pyruvate dehydrogenase complex is one of the highly organized multienzyme system, consisting of multiple copies of three component enzymes, pyruvate dehydrogenase (E1), dihydrolipoyl acetyltransferase (E2), and lipoamide dehydrogenase (E3), which catalyses the irreversible serial reactios specifically and efficiently. The complexes are classified into two types of the central core structures composed of E2s with different symmetries depending on organisms. One has the 432 symmetry in Gram negative bacteria and the other the 532 symmetry in Gram positive bacteria and in eukaryotes. The cyustal structure of the isolated component of E3 from yeast was determined for the latter type. It has been found that there are two types of crystals which are different with each other in crystal habit. The crystal structures were solved by the molecular replacement method. The initial phases were improved by averaging and flattening of electron density map. The atomic coordinates of the molecular model constracted by FRODO were refined by XPLOR with a constrain of non-crystallographic 2-fold symmetry. Although the molecular packings are different, but the molecular structures are similar. Closer comparison with glutathion reductase indicated that although the large atomic deviations are observed in the C-terminal domain, the active site between the two domains has almost the same 3-D structure. This similarity is also observed in E3 from the different organisms with 432 symmetry. From the present investigation, it has been thus revealed that the tertiary structures of E3s are essentially the same even in the different architecture between the two types. It is noticed that such a strong conservation of the structure may be the functional restrain by the enzyme.