Arjunan P, Umland T, Dyda F, Swaminathan S, Furey W, Sax M, Farrenkopf B, Gao Y, Zhang D, Jordan F
Biocrystallography Laboratory, VA Medical Center, Pittsburgh, PA 15240, USA.
J Mol Biol. 1996 Mar 1;256(3):590-600. doi: 10.1006/jmbi.1996.0111.
The crystal structure of pyruvate decarboxylase (EC 4.1.1.1), a thiamin diphosphate-dependent enzyme isolated from Saccharomyces cerevisiae, has been determined and refined to a resolution of 2.3 A. Pyruvate decarboxylase is a homotetrameric enzyme which crystallizes with two subunits in an asymmetric unit. The structure has been refined by a combination of simulated annealing and restrained least squares to an R factor of 0.165 for 46,787 reflections. As in the corresponding enzyme from Saccharomyces uvarum, the homotetrameric holoenzyme assembly has approximate 222 symmetry. In addition to providing more accurate atomic parameters and certainty in the sequence assignments, the high resolution and extensive refinement resulted in the identification of several tightly bound water molecules in key structural positions. These water molecules have low temperature factors and make several hydrogen bonds with protein residues. There are six such water molecules in each cofactor binding site, and one of them is involved in coordination with the required magnesium ion. Another may be involved in the catalytic reaction mechanism. The refined model includes 1074 amino acid residues (two subunits), two thiamin diphosphate cofactors, two magnesium ions associated with cofactor binding and 440 water molecules. From the refined model we conclude that the resting state of the enzyme-cofactor complex is such that the cofactor is already deprotonated at the N4' position of the pyrimidine ring, and is poised to accept a proton from the C2 position of the thiazolium ring.
已确定并精修了从酿酒酵母中分离出的硫胺素二磷酸依赖性酶丙酮酸脱羧酶（EC 4.1.1.1）的晶体结构，分辨率达到2.3埃。丙酮酸脱羧酶是一种同四聚体酶，其不对称单元中含有两个亚基。通过模拟退火和约束最小二乘法相结合，对46,787个反射的结构进行了精修，R因子为0.165。与来自葡萄汁酵母的相应酶一样，同四聚体全酶组装体具有近似的222对称性。除了提供更准确的原子参数和序列归属的确定性外，高分辨率和广泛的精修还导致在关键结构位置鉴定出几个紧密结合的水分子。这些水分子具有低温因子，并与蛋白质残基形成多个氢键。每个辅因子结合位点有六个这样的水分子，其中一个参与与所需镁离子的配位。另一个可能参与催化反应机制。精修后的模型包括1074个氨基酸残基（两个亚基）、两个硫胺素二磷酸辅因子、两个与辅因子结合相关的镁离子和440个水分子。从精修后的模型中我们得出结论，酶 - 辅因子复合物的静止状态使得辅因子在嘧啶环的N4'位置已经去质子化，并准备从噻唑环的C2位置接受一个质子。
