Creation of an Allosteric Phosphofructokinase Starting with a Nonallosteric Enzyme

Abstract

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 M), strong activation by fructose 2,6-bisphosphate (K act ‫؍‬ 0.1 M) and fructose 1,6-bisphosphate (K act ‫؍‬ 40 M), 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 M) 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.

Identification of C-terminal motifs responsible for transmission of inhibition by ATP of mammalian phosphofructokinase, and their contribution to other allosteric effects

“…These results agree with recent findings obtained by mutations of the non-regulatory PFK from Dictyostelium discoideum, which suggested that the action of the inhibitor MgATP is decoupled from Fru-6-P co-operativity, and that stimulation by Fru-2,6-P 2 operates on the same R/T conformational transition that accounts for co-operativity [20]. These results agree with recent findings obtained by mutations of the non-regulatory PFK from Dictyostelium discoideum, which suggested that the action of the inhibitor MgATP is decoupled from Fru-6-P co-operativity, and that stimulation by Fru-2,6-P 2 operates on the same R/T conformational transition that accounts for co-operativity [20].…”

“…Auxiliary enzymes were desalted as described [20]. In all cases, the reaction was started after a 5-min preincubation by the addition of Fru-6-P, and was followed by measuring the absorbance change at 340 nm at 25 • C. When PFK activity was assayed during purification, glucose 6-phosphate was added to Fru-6-P in proportions of 3:1 (mol/mol).…”

Electron microscopy analysis of mammalian phosphofructokinase reveals an unusual 3-dimensional structure with significant implications for enzyme function

“…Human muscle PFK-M and Dictyostelium discoideum PFK were expressed in S. cerevisiae strain HD152-1D carrying deletions in both yeast PFK genes (10), and the recombinant proteins were purified and assayed as described previously (29,30). Protein concentration was determined by the Bradford dye binding method (31), using bovine ␥ globulin as standard.…”

Subunit Interactions and Composition of the Fructose 6-Phosphate Catalytic Site and the Fructose 2,6-Bisphosphate Allosteric Site of Mammalian Phosphofructokinase

“…The effect of Fru-1,6-P 2 was examined as described (27). Auxiliary enzymes were desalted as described (29). Fructose-1,6-bisphosphatase and fructose-2,6-bisphosphatase activities were measured as described by Aragón et al (30), except that the pH was 7.0 and Fru-1,6-P 2 concentration was 50 M, and by González-Mateos et al (31), respectively.…”