A single point mutation leads to an instability of the hetero-octameric structure of yeast phosphofructokinase

Yeast phosphofructokinase is an oligomeric enzyme whose detectable activity in vitro depends on its hetero-octameric structure. Here we provide data d emonstrating that an alanine residue at positions 874 (for the PFK1-encoded alpha-subunit) or 868 (for the PFK2-encoded beta-subunit) is crucial to ac hieve this structure. Thus subunits carrying substitutions by either aspart ate or lysine of this residue cause a lack of phosphofructokinase activity in vitro and signals of the subunits are poorly detectable in Western blots . Size-exclusion HPLC in conjunction with ELISA detection of the enzyme pro tein confirmed that no functional octamer is produced in such mutants. Our data suggest that the mutant subunits., not being assembled, tend to aggreg ate and subsequently become degraded. Substitution of the alanine by valine in either subunit leads to a reduction in specific activities, as expected from a conservative exchange. The kinetic data of the latter mutant reveal ed a higher affinity to the substrate fructose 6-phosphate, a lower extent of ATP inhibition and a lower degree of activation by fructose 2,6-bisphosp hate. In addition, the affinity of mutants carrying a valine instead of an alanine in either the alpha- or the beta-subunit to fructose 2,6-bisphuspha te was increased. As no X-ray data on eukaryotic phosphofructokinases are a vailable yet, our data provide the first evidence that a non-charge amino a cid at position 874 or 868 is essential for the formation of the functional oligomer. This conclusion is substantiated by comparison with the structur e of the well-known prokaryotic enzyme.