Sequence analysis and molecular characterization of Clonorchis sinensis hexokinase, an unusual trimeric 50-kDa glucose-6-phosphate-sensitive allosteric enzyme.
Sequence analysis and molecular characterization of Clonorchis sinensis hexokinase, an unusual trimeric 50-kDa glucose-6-phosphate-sensitive allosteric enzyme.
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华支睾吸虫己糖激酶（一种不寻常的三聚体 50-kDa 葡萄糖-6-磷酸盐敏感性变构酶）的序列分析和分子表征
DOI：
10.1371/journal.pone.0107940
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Yu X
中科院分区：
文献类型：
--
作者：
Chen T;Ning D;Sun H;Li R;Shang M;Li X;Wang X;Chen W;Liang C;Li W;Mao Q;Li Y;Deng C;Wang L;Wu Z;Huang Y;Xu J;Yu X
华支睾吸虫病（Clonorchiasis sinensis，C. sinensis），与胆管癌高度相关。由于现有的检查、治疗和阻断传播提供的预防感染机会有限，因此迫切需要制定预防和控制华支睾吸虫病的综合战略。糖酵解酶是吸虫生存的关键分子，并已成为药物开发的目标。己糖激酶C.对糖酵解途径的第一个关键调控酶CsHK进行了研究。CsHK的计算分子量（Mr）为50.0 kDa。所获得的重组CsHK（rCsHK）是具有约164 kDa的Mr的同源三聚体，如使用非变性PAGE和凝胶过滤所确定的。在pH 10下用50 mM甘氨酸-NaOH和在pH 8.5和10下用100 mM Tris-HCl获得最高活性。rCsHK的动力学具有中等的热稳定性。与相应的阴性对照相比，吡喹酮（PZQ）和抗rCsHK血清可显著抑制酶活性。rCsHK被其底物（包括葡萄糖、甘露糖、果糖和ATP）同型和变构激活。ADP对rCsHK表现出与ATP混合的变构效应，而无机焦磷酸盐（PPi）则表现出与各种变构系统的净变构激活。果糖与底物葡萄糖一起表现为剂量依赖性V激活剂。葡萄糖-6-磷酸（G6 P）对rCsHK的净变构抑制作用与ATP或葡萄糖有关，与各种变构系统呈剂量依赖性。CsHK在成虫、囊蚴、脱囊囊后蚴和虫卵中的mRNA和蛋白水平存在差异。不同发育阶段的能量需求不同。我们的研究进一步了解CsHK的生物学功能，并支持需要筛选CsHK的小分子抑制剂来干扰C.中国的。
Clonorchiasis, which is induced by the infection of Clonorchis sinensis (C. sinensis), is highly associated with cholangiocarcinoma. Because the available examination, treatment and interrupting transmission provide limited opportunities to prevent infection, it is urgent to develop integrated strategies to prevent and control clonorchiasis. Glycolytic enzymes are crucial molecules for trematode survival and have been targeted for drug development. Hexokinase of C. sinensis (CsHK), the first key regulatory enzyme of the glycolytic pathway, was characterized in this study. The calculated molecular mass (Mr) of CsHK was 50.0 kDa. The obtained recombinant CsHK (rCsHK) was a homotrimer with an Mr of approximately 164 kDa, as determined using native PAGE and gel filtration. The highest activity was obtained with 50 mM glycine-NaOH at pH 10 and 100 mM Tris-HCl at pH 8.5 and 10. The kinetics of rCsHK has a moderate thermal stability. Compared to that of the corresponding negative control, the enzymatic activity was significantly inhibited by praziquantel (PZQ) and anti-rCsHK serum. rCsHK was homotropically and allosterically activated by its substrates, including glucose, mannose, fructose, and ATP. ADP exhibited mixed allosteric effect on rCsHK with respect to ATP, while inorganic pyrophosphate (PPi) displayed net allosteric activation with various allosteric systems. Fructose behaved as a dose-dependent V activator with the substrate glucose. Glucose-6-phosphate (G6P) displayed net allosteric inhibition on rCsHK with respect to ATP or glucose with various allosteric systems in a dose-independent manner. There were differences in both mRNA and protein levels of CsHK among the life stages of adult worm, metacercaria, excysted metacercaria and egg of C. sinensis, suggesting different energy requirements during different development stages. Our study furthers the understanding of the biological functions of CsHK and supports the need to screen for small molecule inhibitors of CsHK to interfere with glycolysis in C. sinensis.
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