Chimeric phosphofructokinases involving exchange of the N- and C-terminal halves of mammalian isozymes: implications for ligand binding sites.
Two phosphofructokinase (PFK) chimeras were constructed by exchanging the N- and C-terminal halves of the mammalian M- and C-type isozymes, to investigate the contribution of each terminus to the catalytic site and the fructose-2,6-P(2)/fructose-1,6-P(2) allosteric site. The homogeneously-purified chimeric enzymes organized into tetramers, and exhibited kinetic properties for fructose-6-P and MgATP similar to those of the native enzyme that furnished the N-terminal domain in each case, whereas their fructose-2,6-P(2) activatory characteristics coincided with those of the isozyme that provided the C-terminal half. This reflected the role of each domain in the formation of the corresponding binding site. Grafting the N-terminus of PFK-M onto the C-terminus of the fructose-1,6-P(2) insensitive PFK-C restored transduction of this signal to the catalytic site, which significance is also discussed.
Martínez-Costa
Oscar H
OH
Departamento de Bioquímica de la UAM, Instituto de Investigaciones Biomédicas Alberto Sols UAM-CSIC, Facultad de Medicina de la Universidad Autónoma de Madrid, Arzobispo Morcillo 4, 28029 Madrid, Spain.
Sánchez-Martínez
Cristina
C
Sánchez
Valentina
V
Aragón
Juan J
JJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D005635:Fructosediphosphates / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D025203:Phosphofructokinase-1, Muscle Type / Q000737:chemistry / Q000378:metabolism; D025502:Phosphofructokinase-1, Type C / Q000737:chemistry / Q000378:metabolism; D025221:Phosphofructokinases / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae
D005635:Fructosediphosphates; D007527:Isoenzymes; D008024:Ligands; D011993:Recombinant Fusion Proteins; C027652:fructose 2,6-diphosphate; D000255:Adenosine Triphosphate; D025221:Phosphofructokinases; D025203:Phosphofructokinase-1, Muscle Type; D025502:Phosphofructokinase-1, Type C; C026043:PFKM protein, human; C029063:fructose-1,6-diphosphate