Structural insights into the human and avian IMP cyclohydrolase mechanism via crystal structures with the bound XMP inhibitor.
Structural insights into the human and avian IMP cyclohydrolase mechanism via crystal structures with the bound XMP inhibitor.
复制标题
通过结合 XMP 抑制剂的晶体结构，深入了解人类和鸟类 IMP 环化水解酶机制。
DOI：
10.1021/bi030162i
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Wilson,IanA
中科院分区：
文献类型：
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作者：
Wolan,DennisW;Cheong,Cheom-Gil;Greasley,SamanthaE;Wilson,IanA
在新嘌呤生物合成中，双功能酶ATIC的AICAR转化酶和IMP环水解酶活性将中间的AICAR转化为该途径的最终产物IMP。通过分析禽类ATIC与底物和/或抑制剂复合物的晶体结构，已经确定了AICAR转化酶活性位点并提出了甲酰基转移机制。在此，我们通过比较人类ATIC在1.9 Å分辨率下的XMP抑制剂复合物与先前确定的禽酶的晶体结构，重点研究了IMPCH活性位点和环水解酶机制。我们还确定了第一个人类ATIC结构，以确定在基于结构的抑制剂设计中是否应考虑到与同源禽类酶相比的细微结构差异。这些结构比较，以及与其他IMP和XMP结合蛋白的比较分析，使催化机制得以阐明。IMPCH活性位点的主要作用似乎是诱导底物FAICAR重新配置为能量较低但活性更强的构象。IMPCH活性位点的主链（arg64和Lys66）和侧链相互作用（Thr67）使4-羧酰胺从与AICAR Tfase活性位点结合的首选构象重新定向为促进分子内环化的构象。其他主链酰胺（ile126和Gly127）产生一个氧阴离子孔，帮助定向甲酰基，使其易于被4-羧酰胺胺亲核攻击，然后稳定阴离子中间体。其他几个残基，包括Lys66、Tyr104、Asp125和Lys137 '，提供了底物特异性，并可能通过对酸碱催化的贡献来提高催化速率。
Withinde novopurine biosynthesis, the AICAR transformylase and IMP cyclohydrolase activities of the bifunctional enzyme ATIC convert the intermediate AICAR to the final product of the pathway, IMP. Identification of the AICAR transformylase active site and a proposed formyl transfer mechanism have already resulted from analysis of crystal structures of avian ATIC in complex with substrate and/or inhibitors. Herein, we focus on the IMPCH active site and the cyclohydrolase mechanism through comparison of crystal structures of XMP inhibitor complexes of human ATIC at 1.9 Å resolution with the previously determined avian enzyme. This first human ATIC structure was also determined to ascertain whether any subtle structural differences, compared to the homologous avian enzyme, should be taken into account for structure-based inhibitor design. These structural comparisons, as well as comparative analyses with other IMP and XMP binding proteins, have enabled a catalytic mechanism to be formulated. The primary role of the IMPCH active site appears to be to induce a reconfiguration of the substrate FAICAR to a less energetically favorable, but more reactive, conformer. Backbone (Arg64and Lys66) and side chain interactions (Thr67) in the IMPCH active site reorient the 4-carboxamide from the preferred conformer that binds to the AICAR Tfase active site to one that promotes intramolecular cyclization. Other backbone amides (Ile126and Gly127) create an oxyanion hole that helps orient the formyl group for nucleophilic attack by the 4-carboxamide amine and then stabilize the anionic intermediate. Several other residues, including Lys66, Tyr104, Asp125, and Lys137‘, provide substrate specificity and likely enhance the catalytic rate through contributions to acid−base catalysis.