A MUTANT PHOSPHOFRUCTOKINASE PRODUCES A FUTILE CYCLE DURING GLUCONEOGENESIS IN ESCHERICHIA-COLI

Strains of Escherichia coil bearing different forms of phosphofructoki nase were used to assess the occurrence of futile cycling in cell resu spensions supplied with glycerol as gluconeogenic carbon source. A mod el was used to simulate results of different kinds of experiments for different levels of futile cycle. The main predictions of the model we re experimentally confirmed in a strain with a mutant phosphofructokin ase-2 (phosphofructokinase-2()) which is not inhibited by MgATP, The intracellular fructose 1,6-bisphosphate concentration reaches signific antly 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-bispho sphate, from a trace of [U-C-14]glucose supplied during gluconeogenesi s, indicating higher kinase activity in these conditions. Cell resuspe nsions of the mutant-bearing strain produced higher levels of radioact ively labelled CO2 when supplied with [U-C-14]glycerol as the only car bon source. Simultaneously, fewer glycerol carbons were incorporated i nto HClO4-insoluble macromolecules. Finally, radioactive CO2 output wa s measured in resuspensions supplied with glycerol as the major carbon source with traces of either [1-C-14]glucose or [6-C-14]glucose. It w as found that, whereas in the strains with either of the wild-type pho sphofructokinase isoenzymes, radioactive CO2 output from [1-C-14]gluco se was higher than with [6-C-14]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 r ates predicted by the model, an explanation linking the futile cycle t o the differential labelling of CO2 is advanced, Finally, on the basis of these results it is proposed that strains bearing phosphofructokin ase-2() sustain higher rates of futile cycling during gluconeogenesis than strains bearing either of the wildtype isoforms of phosphofructo kinase. 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 Cent re, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom cop ies can be obtained on the terms indicated in Biochem. J. (1997) 321, 8.