Single point mutations in either gene encoding the subunits of the heterooctameric yeast phosphofructokinase abolish allosteric inhibition by ATP

Yeast phosphofructokinase is a heterooctameric enzyme subject to a complex allosteric regulation. A mutation in the PFK1 gene, encoding the larger cy- subunits, rendering the enzyme insensitive to allosteric inhibition by ATP was found to be caused by an exchange of proline 728 for a leucine residue. By in vitro mutagenesis, we introduced this mutation in either PFK1 or PFK 2 and found that the exchange in either subunit drastically reduced the sen sitivity of the holoenzyme to ATP inhibition. This was accompanied by a lac k of allosteric activation by AMP, fructose a,6-bisphosphate, or ammonium a nd an increased resistance to heat inactivation. Yeast cells carrying eithe r one mutation or both in conjunction did not display a strong phenotype wh en grown on fermentable carbon sources and did not show any significant cha nges in intermediary metabolites. Growth on non-fermentable carbon sources was clearly impaired. The strain carrying both mutant alleles was more sens itive to Congo Red than the wildtype strain or the single mutants indicatin g differences in cell wall composition. In addition, we found single pfk nu ll mutants to be less viable than wild type at different storage temperatur es and a pfk2 null mutant to be temperature-sensitive for growth at 37 degr eesC. The latter mutant was shown to be respiration-dependent for growth on glucose.