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 demonstrating that an alanine residue at positions 874 (for the PFK1-encoded alpha-subunit) or 868 (for the PFK2-encoded beta-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 alpha- or the beta-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.
Kirchberger
J
J
Institut für Biochemie der Medizinischen Fakultät der Universität Leipzig, Liebigstrasse 16, D-04103 Leipzig, Germany.
Edelmann
A
A
Kopperschläger
G
G
Heinisch
J J
JJ
D000409:Alanine / Q000235:genetics; D000483:Alleles; D000595:Amino Acid Sequence; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D005800:Genes, Fungal; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016296:Mutagenesis; D009154:Mutation; D010732:Phosphofructokinase-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014633:Valine / Q000235:genetics
D007527:Isoenzymes; D010732:Phosphofructokinase-1; D014633:Valine; D000409:Alanine