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首页> 外文期刊>Biochemistry >A closer look at the active site of gamma-class carbonic anhydrases: high-resolution crystallographic studies of the carbonic anhydrase from Methanosarcina thermophila.
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A closer look at the active site of gamma-class carbonic anhydrases: high-resolution crystallographic studies of the carbonic anhydrase from Methanosarcina thermophila.

机译：仔细研究伽玛级碳酸酐酶的活性位点：嗜热甲烷菌的碳酸酐酶的高分辨率晶体学研究。

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摘要

The prototype of the gamma-class of carbonic anhydrase has been characterized from the methanogenic archaeon Methanosarcina thermophila. Previously reported kinetic studies of the gamma-class carbonic anhydrase are consistent with this enzyme having a reaction mechanism similar to that of the mammalian alpha-class carbonic anhydrase. However, the overall folds of these two enzymes are dissimilar, and apart from the zinc-coordinating histidines, the active site residues bear little resemblance to one another. The crystal structures of zinc-containing and cobalt-substituted gamma-class carbonic anhydrases from M. thermophila are reported here between 1.46 and 1.95 A resolution in the unbound form and cocrystallized with either SO(4)(2)(-) or HCO(3)(-). Relative to the tetrahedral coordination geometry seen at the active site in the alpha-class of carbonic anhydrases, the active site of the gamma-class enzyme contains additional metal-bound water ligands, so the overall coordination geometry is trigonal bipyramidal for the zinc-containing enzyme and octahedral for the cobalt-substituted enzyme. Ligands bound to the active site all make contacts with the side chain of Glu 62 in manners that suggest the side chain is likely protonated. In the uncomplexed zinc-containing enzyme, the side chains of Glu 62 and Glu 84 appear to share a proton; additionally, Glu 84 exhibits multiple conformations. This suggests that Glu 84 may act as a proton shuttle, which is an important aspect of the reaction mechanism of alpha-class carbonic anhydrases. A hydrophobic pocket on the surface of the enzyme may participate in the trapping of CO(2) at the active site. On the basis of the coordination geometry at the active site, ligand binding modes, the behavior of the side chains of Glu 62 and Glu 84, and analogies to the well-characterized alpha-class of carbonic anhydrases, a more-defined reaction mechanism is proposed for the gamma-class of carbonic anhydrases.
机译：γ-级碳酸酐酶的原型已从产甲烷的古细菌甲烷八叠球菌（Methanosarcina thermophila）进行了表征。先前报道的对γ-类碳酸酐酶的动力学研究与该酶具有类似于哺乳动物α-类碳酸酐酶的反应机理相一致。然而，这两种酶的总折叠是不同的，并且除了锌配位组氨酸之外，活性位点残基彼此之间几乎没有相似之处。此处报道了嗜热毁丝霉的含锌和钴取代的γ-类碳酸酐酶的晶体结构在1.46和1.95 A之间的未结合形式并与SO（4）（2）（-）或HCO（ 3）（-）。相对于碳酸酐酶的α类活性位点上看到的四面体配位几何形状，γ类酶的活性位点包含其他与金属结合的水配体，因此总体配位几何形状为含锌的三角锥型酶和八面体为钴取代的酶。结合至活性位点的配体均以暗示侧链可能质子化的方式与Glu 62的侧链接触。在未络合的含锌酶中，Glu 62和Glu 84的侧链似乎共享一个质子。另外，Glu 84表现出多种构象。这表明Glu 84可以充当质子穿梭，这是α级碳酸酐酶反应机理的重要方面。酶表面上的疏水口袋可能参与在活性位点捕获CO（2）。根据活性位点的配位几何构型，配体结合模式，Glu 62和Glu 84侧链的行为以及与碳酸酐酶的特征明确的α类相似的现象，一种更明确的反应机理是被提议用于伽马级的碳酸酐酶。

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