Experimental and in silico analyses of glycolytic flux control in bloodstream form Trypanosoma brucei
Experimental and in silico analyses of glycolytic flux control in bloodstream form Trypanosoma brucei
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DOI：
10.1074/jbc.m502403200
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发表时间：
2005-08-05
影响因子：
4.8
通讯作者：
Michels, PAM
中科院分区：
文献类型：
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作者：
Albert, MA;Haanstra, JR;Michels, PAM
先前基于所有可用的酶动力学数据开发了血流形式布氏锥虫中糖酵解的数学模型（Bakker，B. M.，Michels，P. A. M.，Opperdoes，F. R.，Westerhoff，H. V.（1997）J.Biol.Chem.272，3207 - 3215）。该模型正确预测的流量和细胞代谢物浓度的测量在非生长锥虫和质膜葡萄糖转运蛋白施加的流量控制的主要贡献。令人惊讶的是，预测了己糖激酶（HXK）、磷酸果糖激酶（PFK）和丙酮酸激酶（PYK）的大的产能过剩。在这里，我们提出了我们的进一步分析的控制糖酵解流量的血流形式T。布鲁塞。首先，该模型进行了优化和扩展与最近的信息酶的动力学和它们的活动，在体外培养的生长锥虫的裂解物中测量。第二，通过RNA干扰改变锥虫中五种糖酵解酶（HXK、PFK、磷酸甘油酸酯酶、烯醇化酶和PYK）的浓度。敲低这些酶的生长，活动和各种酶的水平和糖酵解通量的影响进行了研究，并与模型预测进行了比较。由此获得的数据支持计算机模拟分析的结论，即HXK、PFK和PYK过量，尽管低于预测值。有趣的是，PFK和烯醇化酶的消耗对一些其他糖酵解酶的活性（但不影响或在较小程度上影响表达）有影响。位于糖体（过氧化物酶体样细胞器，含有锥虫糖酵解途径的前七种酶）和细胞质中的酶都受到影响。这些数据表明，锥虫糖酵解中存在新的调节机制。
A mathematical model of glycolysis in bloodstream form Trypanosoma brucei was developed previously on the basis of all available enzyme kinetic data (Bakker, B. M., Michels, P. A. M., Opperdoes, F. R., and Westerhoff, H. V. (1997) J. Biol. Chem. 272, 3207 - 3215). The model predicted correctly the fluxes and cellular metabolite concentrations as measured in non-growing trypanosomes and the major contribution to the flux control exerted by the plasma membrane glucose transporter. Surprisingly, a large overcapacity was predicted for hexokinase (HXK), phosphofructokinase (PFK), and pyruvate kinase (PYK). Here, we present our further analysis of the control of glycolytic flux in bloodstream form T. brucei. First, the model was optimized and extended with recent information about the kinetics of enzymes and their activities as measured in lysates of in vitro cultured growing trypanosomes. Second, the concentrations of five glycolytic enzymes (HXK, PFK, phosphoglycerate mutase, enolase, and PYK) in trypanosomes were changed by RNA interference. The effects of the knockdown of these enzymes on the growth, activities, and levels of various enzymes and glycolytic flux were studied and compared with model predictions. Data thus obtained support the conclusion from the in silico analysis that HXK, PFK, and PYK are in excess, albeit less than predicted. Interestingly, depletion of PFK and enolase had an effect on the activity ( but not, or to a lesser extent, expression) of some other glycolytic enzymes. Enzymes located both in the glycosomes ( the peroxisome-like organelles harboring the first seven enzymes of the glycolytic pathway of trypanosomes) and in the cytosol were affected. These data suggest the existence of novel regulatory mechanisms operating in trypanosome glycolysis.