title: Distinct functional roles of the two terminal halves of eukaryotic phosphofructokinase.
abstract: Eukaryotic PFK (phosphofructokinase), a key regulatory enzyme in glycolysis, has homologous N- and C-terminal domains thought to result from duplication, fusion and divergence of an ancestral prokaryotic gene. It has been suggested that both the active site and the Fru-2,6-P2 (fructose 2,6-bisphosphate) allosteric site are formed by opposing N- and C-termini of subunits orientated antiparallel in a dimer. In contrast, we show in the present study that in fact the N-terminal halves form the active site, since expression of the N-terminal half of the enzymes from Dictyostelium discoideum and human muscle in PFK-deficient yeast restored growth on glucose. However, the N-terminus alone was not stable in vitro. The C-terminus is not catalytic, but is needed for stability of the enzyme, as is the connecting peptide that normally joins the two domains (here included in the N-terminus). Co-expression of homologous, but not heterologous, N- and C-termini yielded stable fully active enzymes in vitro with sizes and kinetic properties similar to those of the wild-type tetrameric enzymes. This indicates that the separately translated domains can fold sufficiently well to bind to each other, that such binding of complementary domains is stable and that the alignment is sufficiently accurate and tight as to preserve metabolite binding sites and allosteric interactions.
authors.affiliation: Departamento de Bioquímica de la Universidad Autónoma de Madrid and Instituto de Investigaciones Biomédicas Alberto Sols Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas, Facultad de Medicina de la Universidad Autónoma de Madrid, 28029 Madrid, Spain. oscar.martinez@uam.es
authors.forename: Oscar H
authors.initials: OH
authors.lastname: Martínez-Costa
authors.forename: Valentina
authors.initials: V
authors.lastname: Sánchez
authors.forename: Antonio
authors.initials: A
authors.lastname: Lázaro
authors.forename: Eloy D
authors.initials: ED
authors.lastname: Hernández
authors.forename: Keith
authors.initials: K
authors.lastname: Tornheim
authors.forename: Juan J
authors.initials: JJ
authors.lastname: Aragón
mesh_terms: D000495:Allosteric Site; D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D004023:Dictyostelium / Q000201:enzymology*; D056890:Eukaryota; D005635:Fructosediphosphates / Q000378:metabolism; D006019:Glycolysis; D006801:Humans; D015151:Immunoblotting; D009132:Muscles / Q000201:enzymology*; D050506:Mutant Chimeric Proteins / Q000235:genetics / Q000378:metabolism*; D025221:Phosphofructokinases / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
chemical_list: D005635:Fructosediphosphates; D050506:Mutant Chimeric Proteins; D011993:Recombinant Fusion Proteins; C027652:fructose 2,6-diphosphate; D025221:Phosphofructokinases