Optimum activity of the phosphofructokinase from Ascaris suum requires more than one metal ion.
Phosphofructokinase (PFK) catalyzes the phosphorylation of fructose 6-phosphate (F6P) to give fructose 1,6-bisphosphate (FBP) using MgATP as the phosphoryl donor. As the concentration of Mg(2+) increases above the concentration needed to generate the MgATP chelate complex, a 15-fold increase in the initial rate was observed at low MgATP. The effect of Mg(2+) is limited to V/K(MgATP), and initial rate studies indicate an equilibrium-ordered addition of Mg(2+) before MgATP. Isotope partitioning of the dPFK:MgATP complex indicates a random addition of MgATP and F6P at low Mg(2+), with the rate of release of MgATP from the central E:MgATP:F6P complex 4-fold faster than the net rate constant for catalysis. This can be contrasted with the ordered addition of MgATP prior to F6P at high Mg(2+). The addition of fructose 2,6-bisphosphate (F26P(2)) has no effect on the mechanism at low Mg(2+), with the exception of a 4-fold increase in the affinity of the enzyme for F6P. At high Mg(2+), F26P(2) causes the kinetic mechanism to become random with respect to MgATP and F6P and with MgATP released from the central complex half as fast as the net rate constant for catalysis. The latter is in agreement with previous studies [Gibson, G. E., Harris, B. G., and Cook, P. F. (1996) Biochemistry 35, 5451-5457]. The overall effect of Mg(2+) is a decrease in the rate of release of MgATP from the E:MgATP:F6P complex, independent of the concentration of F26P(2).
Department of Molecular Biology and Immunology, University of North Texas Health Science Center at Fort Worth, 3500 Camp Bowie Boulevard, Fort Worth, Texas 76107, USA.
Grant E
GE
Gibson
Ben G
BG
Harris
Paul F
PF
Cook
D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D017165:Ascaris suum / Q000201:enzymology*; D004047:Diethyl Pyrocarbonate / Q000737:chemistry; D004789:Enzyme Activation; D005635:Fructosediphosphates / Q000737:chemistry; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry*; D025221:Phosphofructokinases / Q000737:chemistry / Q000378:metabolism*
D005635:Fructosediphosphates; C027652:fructose 2,6-diphosphate; D000255:Adenosine Triphosphate; D025221:Phosphofructokinases; D008274:Magnesium; D004047:Diethyl Pyrocarbonate