Structural characterization of the enzyme-substrate, enzyme-intermediate, and enzyme-product complexes of thiamin phosphate synthase
Structural characterization of the enzyme-substrate, enzyme-intermediate, and enzyme-product complexes of thiamin phosphate synthase
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DOI：
10.1021/bi0104726
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发表时间：
2001-08-28
期刊：
影响因子：
2.9
通讯作者：
Ealick, SE
中科院分区：
文献类型：
--
作者：
Peapus, DH;Chiu, HJ;Ealick, SE
磷酸硫胺素合酶催化4-氨基-5(羟甲基)-2-甲基嘧啶焦磷酸和5-(羟乙基)-4-甲基噻唑磷酸形成磷酸硫胺素。多项证据表明该反应通过解离机制进行。先前确定的磷酸硫胺素合酶与反应产物磷酸硫胺素和焦磷酸镁复合物的晶体结构提供了活性位点的视图并建议了许多额外的实验。我们在此报告了七种新的晶体结构，主要涉及浸泡在含有底物或产物的溶液中的 S130A 硫胺素磷酸合酶晶体。我们制备了 S130A 硫胺素磷酸合酶，旨在表征酶-底物复合物。令人惊讶的是，在三种硫胺素磷酸合酶结构中，活性位点密度不能建模为底物或产物。对于这些结构，电子密度的最佳拟合是由独立的嘧啶、焦磷酸盐和噻唑磷酸盐片段组成的模型提供的，与碳正离子中间体一致。所得的碳正离子可能通过质子从嘧啶氨基转移到焦磷酸盐而进一步稳定，得到嘧啶亚胺甲基化物，我们认为这是在晶体结构中观察到的物质。
Thiamin phosphate synthase catalyzes the formation of thiamin phosphate from 4-amino-5(hydroxymethyl)-2-methylpyrimidine pyrophosphate and 5-(hydroxyethyl)-4-methylthiazole phosphate. Several lines of evidence suggest that the reaction proceeds via a dissociative mechanism. The previously determined crystal structure of thiamin phosphate synthase in complex with the reaction products, thiamin phosphate and magnesium pyrophosphate, provided a view of the active site and suggested a number of additional experiments. We report here seven new crystal structures primarily involving crystals of S130A thiamin phosphate synthase soaked in solutions containing substrates or products. We prepared S130A thiamin phosphate synthase with the intent of characterizing the enzyme-substrate complex. Surprisingly, in three thiamin phosphate synthase structures, the active site density cannot be modeled as either substrates or products. For these structures, the best fit to the electron density is provided by a model that consists of independent pyrimidine, pyrophosphate, and thiazole phosphate fragments, consistent with a carbenium ion intermediate. The resulting carbenium ion is likely to be further stabilized by proton transfer from the pyrimidine amino group to the pyrophosphate to give the pyrimidine iminemethide, which we believe is the species that is observed in the crystal structures.