Structure and inhibition of orotidine 5'-monophosphate decarboxylase from Plasmodium falciparum
Structure and inhibition of orotidine 5'-monophosphate decarboxylase from Plasmodium falciparum
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DOI：
10.1021/bi702390k
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发表时间：
2008-03-25
期刊：
影响因子：
2.9
通讯作者：
Christopherson, Richard I.
中科院分区：
文献类型：
--
作者：
Langley, David B.;Shojaei, Maryam;Christopherson, Richard I.
恶性疟原虫Orotidine 5’- monophospate （OMP）脱羧酶（PfODCase, EC 4.1.1.23）的过表达、纯化、动力学和生化分析以及结晶。天然酶为同二聚体，亚基分子质量为38 kDa。底物OMP的饱和曲线符合Michaelis-Menten动力学，Km = 350 +/- 60 nM, V-max = 2.70 +/- 0.10 μ mol/min/mg蛋白。5′-单磷酸核苷类似物的抑制模式与OMP呈线性竞争关系，对PfODCase的抑制效果依次递减：吡唑呋喃5′-单磷酸（K-i = 3.6 +/- 0.7 nM）、5′-单磷酸黄嘌呤5′-单磷酸（XMP, K-i = 4.4 +/- 0.7 nM）、6-偶氮吡啶5′-单磷酸（AzaUMP, K-i = 12 +/- 3 nM）、4 -别嘌呤醇-3-核苷5′-单磷酸（Ki = 240 +/- 20 nM）。XMP对PfODCase的抑制作用是人酶的150倍。在没有配体的情况下，解出了PfODCase的结构，并表现出典型的tim -桶状折叠特征。磷酸结合环和α - 5环都具有构象柔韧性，这可能与反应途径中底物捕获和产物释放有关。
Orotidine 5'-monophosphate (OMP) decarboxylase from Plasmodium falciparum (PfODCase, EC 4.1.1.23) has been overexpressed, purified, subjected to kinetic and biochemical analysis, and crystallized. The native enzyme is a homodimer with a subunit molecular mass of 38 kDa. The saturation curve for OMP as a substrate conformed to Michaelis-Menten kinetics with Km = 350 +/- 60 nM and V-max = 2.70 +/- 0.10 mu mol/min/mg protein. Inhibition patterns for nucleoside 5'-monophosphate analogues were linear competitive with respect to OMP with a decreasing potency of inhibition of PfODCase in the order: pyrazofurin 5'-monophosphate (K-i = 3.6 +/- 0.7 nM) > xanthosine 5'-monophosphate (XMP, K-i = 4.4 +/- 0.7 nM) > 6-azauridine 5'-monophosphate (AzaUMP, K-i = 12 +/- 3 nM) > allopurinol-3-riboside 5'-monophosphate (Ki = 240 20 nM). XMP is an similar to 150-fold more potent inhibitor of PfODCase compared with the human enzyme. The structure of PfODCase was solved in the absence of ligand and displays a classic TIM-barrel fold characteristic of the enzyme. Both the phosphate-binding loop and the beta alpha 5-loop have conformational flexibility, which may be associated with substrate capture and product release along the reaction pathway.