Observation of covalent intermediates in an enzyme mechanism at atomic resolution
Observation of covalent intermediates in an enzyme mechanism at atomic resolution
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DOI：
10.1126/science.1063601
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发表时间：
2001-10-12
期刊：
影响因子：
56.9
通讯作者：
Wilson, IA
中科院分区：
文献类型：
--
作者：
Heine, A;DeSantis, G;Wilson, IA
在经典酶学中，催化机制中的中间体和过渡态通常是从一系列生化实验中推断出来的。在这里，我们从反应中间体的真实原子分辨率 X 射线结构中推导出酶机制。野生型和突变型 D-2-脱氧核糖-5-磷酸 (DRP) 醛缩酶复合物的两种超高分辨率结构（DRP 为 1.05 和 1.10 埃）明确识别了醛缩酶机制中假定的共价甲醇胺和席夫碱中间体。结合定点诱变和 H-1 核磁共振，我们现在可以提出迄今为止难以捉摸的 C-2 质子提取步骤和整个立体化学过程是如何完成的。质子中继系统似乎可以激活保守的活性位点水，该水充当质子转移的关键介体。
In classical enzymology, intermediates and transition states in a catalytic mechanism are usually inferred from a series of biochemical experiments. Here, we derive an enzyme mechanism from true atomic-resolution x-ray structures of reaction intermediates. Two ultra-high resolution structures of wild-type and mutant D-2-deoxyribose-5-phosphate (DRP) aldolase complexes with DRP at 1.05 and 1.10 angstroms unambiguously identify the postulated covalent carbinolamine and Schiff base intermediates in the aldolase mechanism. In combination with site-directed mutagenesis and H-1 nuclear magnetic resonance, we can now propose how the heretofore elusive C-2 proton abstraction step and the overall stereochemical course are accomplished. A proton relay system appears to activate a conserved active-site water that functions as the critical mediator for proton transfer.