Creation of an allosteric phosphofructokinase starting with a nonallosteric enzyme. The case of dictyostelium discoideum phosphofructokinase.
An allosteric phosphofructokinase (PFK) was created by sequence manipulation of the nonallosteric enzyme from the slime mold Dictyostelium discoideum (DdPFK). Most amino acid residues proposed as important for catalytic and allosteric sites are conserved in DdPFK except for a few of them, and their reversion did not modify its kinetic behavior. However, deletions at the unique C-terminal extension of this PFK produced a markedly allosteric enzyme. Thus, a mutant lacking the last 26 C-terminal residues exhibited hysteresis in the time course, intense cooperativity (n(H) = 3.8), and a 200-fold decrease in the apparent affinity for fructose 6-phosphate (S(0.5) = 4500 microm), strong activation by fructose 2,6-bisphosphate (K(act) = 0.1 microm) and fructose 1,6-bisphosphate (K(act) = 40 microm), dependence on enzyme concentration, proton inhibition, and subunit association-dissociation in response to fructose 6-phosphate versus the nonhysteretic and hyperbolic wild-type enzyme (n(H) = 1.0; K(m) = 22 microm) that remained as a stable tetramer. Systematic deletions and point mutations at the C-tail region of DdPFK identified the last C-terminal residue, Leu(834), as critical to produce a nonallosteric enzyme. All allosteric mutants were practically insensitive to MgATP inhibition, suggesting that this effect does not involve the same allosteric transition as that responsible for fructose 6-phosphate cooperativity and fructose bisphosphate activation.
Santamaria
Belen
B
Departamento de Bioquimica and Instituto de Investigaciones Biomédicas Alberto Sols UAM-CSIC, Facultad de Medicina de la Universidad Autónoma, 28029 Madrid, Spain.
Estevez
Antonio M
AM
Martinez-Costa
Oscar H
OH
Aragon
Juan J
JJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D000494:Allosteric Regulation; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002845:Chromatography; D004023:Dictyostelium / Q000201:enzymology* / Q000235:genetics; D005635:Fructosediphosphates / Q000378:metabolism; D005636:Fructosephosphates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D025221:Phosphofructokinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000378:metabolism; D015202:Protein Engineering; D016415:Sequence Alignment; D012997:Solvents / Q000378:metabolism; D013997:Time Factors
D005635:Fructosediphosphates; D005636:Fructosephosphates; D012997:Solvents; D011092:Polyethylene Glycols; C027618:fructose-6-phosphate; C027652:fructose 2,6-diphosphate; D000255:Adenosine Triphosphate; D025221:Phosphofructokinases