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首页> 外文期刊>The FEBS journal >Structural and biochemical characterization of the folyl-poly-gamma-L-glutamate hydrolyzing activity of human glutamate carboxypeptidase II
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Structural and biochemical characterization of the folyl-poly-gamma-L-glutamate hydrolyzing activity of human glutamate carboxypeptidase II

机译：人谷氨酸羧肽酶II的叶酰-聚-γ-L-谷氨酸水解活性的结构和生化特性

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摘要

In addition to its well-characterized role in the central nervous system, human glutamate carboxypeptidase II (GCPII; Uniprot ID Q04609) acts as a folate hydrolase in the small intestine, participating in the absorption of dietary polyglutamylated folates (folyl-n-gamma-L-glutamic acid), which are the provitamin form of folic acid (also known as vitamin B-9). Despite the role of GCPII as a folate hydrolase, nothing is known about the processing of polyglutamylated folates by GCPII at the structural or enzymological level. Moreover, many epidemiologic studies on the relationship of the naturally occurring His475Tyr polymorphism to folic acid status suggest that this polymorphism may be associated with several pathologies linked to impaired folate metabolism. In the present study, we report: (a) a series X-ray structures of complexes between a catalytically inactive GCPII mutant (Glu424Ala) and a panel of naturally occurring polyglutamylated folates; (b) the X-ray structure of the His475Tyr variant at a resolution of 1.83 angstrom; (c) the study of the recently identified arene-binding site of GCPII through mutagenesis (Arg463Leu, Arg511Leu and Trp541Ala), inhibitor binding and enzyme kinetics with polyglutamylated folates as substrates; and (d) a comparison of the thermal stabilities and folate-hydrolyzing activities of GCPII wild-type and His475Tyr variants. As a result, the crystallographic data reveal considerable details about the binding mode of polyglutamylated folates to GCPII, especially the engagement of the arene binding site in recognizing the folic acid moiety. Additionally, the combined structural and kinetic data suggest that GCPII wild-type and His475Tyr variant are functionally identical.
机译：人谷氨酸羧肽酶II（GCPII; Uniprot ID Q04609）除了在中枢神经系统中具有很好的作用外，还作为小肠中的叶酸水解酶，参与了膳食中聚谷氨酰化叶酸（folyl-n-γ- L-谷氨酸），是叶酸的原维生素形式（也称为维生素B-9）。尽管GCPII作为叶酸水解酶发挥了作用，但关于GCPII在结构或酶学层面对多谷氨酰化叶酸的加工知之甚少。此外，许多关于自然发生的His475Tyr多态性与叶酸状态之间关系的流行病学研究表明，这种多态性可能与叶酸代谢受损相关的几种病理相关。在本研究中，我们报告：（a）催化失活的GCPII突变体（Glu424Ala）与一组天然存在的聚谷氨酰叶酸之间的复合物的一系列X射线结构; （b）His475Tyr变体的X射线结构，分辨率为1.83埃; （c）通过诱变（Arg463Leu，Arg511Leu和Trp541Ala），以聚谷氨酰化叶酸为底物的抑制剂结合和酶动力学研究最近确定的GCPII的芳烃结合位点; （d）比较GCPII野生型和His475Tyr变种的热稳定性和叶酸水解活性。结果，晶体学数据揭示了关于聚谷氨酰化叶酸与GCPII的结合模式的大量细节，尤其是芳烃结合位点在识别叶酸部分中的参与。此外，组合的结构和动力学数据表明，GCPII野生型和His475Tyr变体功能相同。

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