A mutant phosphofructokinase produces a futile cycle during gluconeogenesis in Escherichia coli.
A mutant phosphofructokinase produces a futile cycle during gluconeogenesis in Escherichia coli.
复制标题
突变型磷酸果糖激酶在大肠杆菌糖异生过程中产生无效循环。
作者：
J. C. Torres;V. Guixé;J. Babul
使用带有不同形式的磷酸果糖激酶的大肠杆菌菌株来评估在以甘油作为产酶碳源供应的细胞重悬液中无效循环的发生。模型被用来模拟不同类型的实验结果为不同水平的无效循环。该模型的主要预测实验证实了一个菌株的突变体磷酸果糖激酶-2（phosphofuctokinase-2*），这是不受MgATP抑制。细胞内果糖1，6-二磷酸浓度达到显着更高的水平，在携带突变株比在菌株与磷酸果糖激酶-1或-2。此外，该菌株显示出较高的速率和水平的果糖1，6-二磷酸的体内放射性标记，从痕量的[U-14 C]葡萄糖供应在异源发生，表明较高的激酶活性在这些条件下。当提供[U-14 C]甘油作为唯一的碳源时，携带mu的菌株的细胞再悬浮液产生更高水平的放射性标记的CO2。同时，更少的甘油碳被纳入HClO 4不溶性大分子。最后，在以甘油作为主要碳源并含有痕量[1- 14 C]葡萄糖或[6- 14 C]葡萄糖的重悬液中测量放射性CO2输出。结果发现，在具有任一种野生型磷酸果糖激酶同工酶的菌株中，[1- 14 C]葡萄糖的放射性CO2输出高于[6- 14 C]葡萄糖，而具有磷酸果糖激酶-2 * 的菌株则相反。这一结果也与模型的相应预测相一致。使用该模型预测的放射性通量率，解释无效循环的二氧化碳的差异标记先进。最后，在这些结果的基础上，它提出，轴承磷酸果糖激酶-2 * 菌株维持较高的无效循环率比轴承的磷酸果糖激酶的野生型亚型的菌株在异源过程中。用于模型模拟的动力学方程和参数值在补充出版物50183（8页）中给出，该出版物已保藏在英国图书馆文献供应中心，Boston Spa，Wetherby，West约克郡LS 23 7 BQ，U.K.，可以根据Biochem. J.（1997）321，8中所述的条件从其获得拷贝。
Strains of Escherichia coli bearing different forms of phosphofructokinase were used to assess the occurrence of futile cycling in cell resuspensions supplied with glycerol as gluconeogenic carbon source. A model was used to simulate results of different kinds of experiments for different levels of futile cycle. The main predictions of the model were experimentally confirmed in a strain with a mutant phosphofructokinase-2 (phosphofructokinase-2*) which is not inhibited by MgATP. The intracellular fructose 1, 6-bisphosphate concentration reaches significantly higher levels in the mutant-bearing strain than in strains with either phosphofructokinase-1 or -2. Also, this strain showed a higher rate and level of in vivo radioactive labelling of fructose 1, 6-bisphosphate, from a trace of [U-14C]glucose supplied during gluconeogenesis, indicating higher kinase activity in these conditions. Cell resuspensions of the mutant-bearing strain produced higher levels of radioactively labelled CO2 when supplied with [U-14C]glycerol as the only carbon source. Simultaneously, fewer glycerol carbons were incorporated into HClO4-insoluble macromolecules. Finally, radioactive CO2 output was measured in resuspensions supplied with glycerol as the major carbon source with traces of either [1-14C]glucose or [6-14C]glucose. It was found that, whereas in the strains with either of the wild-type phosphofructokinase isoenzymes, radioactive CO2 output from [1-14C]glucose was higher than with [6-14C]glucose, the reverse is found for the strain with phosphofructokinase-2*. This result also agrees with the corresponding prediction of the model. Using the radioactivity flux rates predicted by the model, an explanation linking the futile cycle to the differential labelling of CO2 is advanced. Finally, on the basis of these results it is proposed that strains bearing phosphofructokinase-2* sustain higher rates of futile cycling during gluconeogenesis than strains bearing either of the wild-type isoforms of phosphofructokinase. The kinetic equations and parameter values used for the model simulations are given in Supplementary Publication SUP 50183 (8 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1997) 321, 8.