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

Abstract

Yeast phosphofructokinase is an oligomeric enzyme whose detectable activity in vitro depends on its hetero-octameric structure. Here we provide data demonstrating that an alanine residue at positions 874 (for the PFK1-encoded α-subunit) or 868 (for the PFK2-encoded β-subunit) is crucial to achieve this structure. Thus subunits carrying substitutions by either aspartate 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 protein confirmed that no functional octamer is produced in such mutants. Our data suggest that the mutant subunits, not being assembled, tend to aggregate 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 revealed 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-bisphosphate. In addition, the affinity of mutants carrying a valine instead of an alanine in either the α- or the β-subunit to fructose 2,6-bisphosphate was increased. As no X-ray data on eukaryotic phosphofructokinases are available yet, our data provide the first evidence that a non-charge amino acid at position 874 or 868 is essential for the formation of the functional oligomer. This conclusion is substantiated by comparison with the structure of the well-known prokaryotic enzyme.

Progress in Metabolic Engineering of Saccharomyces cerevisiae

“…For example, research on yeast phosphofructokinase identified mutations that modified allosteric regulation (116,167,296). Nonetheless, there are only a few recent examples of yeast metabolic engineering that take advantage of site-specific enzyme mutagenesis to modify regulatory properties.…”

Single Point Mutations in Either Gene Encoding the Subunits of the Heterooctameric Yeast Phosphofructokinase Abolish Allosteric Inhibition by ATP

“…It is also likely that the Pfk produced in the mutants is still a heterooctamer, since a loss of the quaternary structure in the yeast enzyme usually results in aggregation and degradation of the subunits, leading to a drastic decrease in Western blot signals (see Ref. 43, and references therein), which is not observed in the PFKatp mutants. Given these data, it is not surprising that the mutant strains did not show any obvious defect for growth on glucose media or in respect to glycolytic flux.…”

Saccharomyces cerevisiae RNA Polymerase II Is Affected by Kluyveromyces lactis Zymocin

“…Identical amounts of total protein obtained from three independent experiments of untreated and zymocin-arrested cells were subjected to SDS-PAGE analysis using 6% gels, electroblotted onto polyvinylidene difluoride membranes (Immobilon), and probed with mouse monclonal anti-pol IIA and anti-pol II0 antibodies 8WG16 and H14 (Covance). Protein loadings were compared using a polyclonal rabbit antibody directed against the ␣-and ␤-subunits of yeast Pfk1p (33). pol II quantitation used the Image Master 2D (Amersham Biosciences) program.…”

“…Protein loading was followed using a polyclonal antiserum specific for the ␣-and ␤-subunits of phosphofructokinase 1 (Pfk1p) (33). B, identical protein amounts obtained from cell fractionation of untreated cells and cells arrested with zymocin for 3 h were subjected to 6% SDS-PAGE and immunoprobed with antibodies specific for IIA and II0 (see A).…”