Kinetic studies of fructose 6-phosphate,2-kinase and fructose 2,6-bisphosphatase.
The reactions catalyzed by a bifunctional enzyme, fructose-6-P,2-kinase and fructose 2,6- bisophosphatase , were studied. Fructose-6-P,2-kinase catalyzes the isotope exchange of ADP with ATP in the absence of fructose-6-P at about 3% of the rate of the overall reaction. The Km values for ATP and ADP for the reaction are 120 and 66 microM, respectively. No isotope exchange between fructose-6-P with fructose-2,6-P2 in the absence of adenine nucleotide was observed. The ADP-ATP exchange is not inhibited by the presence of 25 microM (1.6 X Km) fructose-6-P and less than 20% inhibition was observed at higher concentrations. Fructose-6-P,2-kinase catalyzes the reversal of the reaction at a rate approximately one-half that of the forward direction. The fructose 2,6-bisphosphatase reaction is not inhibited by 1 to 20 mM ADP. Plots of the reciprocal of the concentration of either substrate versus the reciprocal of the concentration of the other substrate yield intersecting lines for both forward and reverse reactions. The Michaelis constants for ATP and fructose-6-P are 150 and 16 microM, respectively, while those values for ADP and fructose-2,6-P2 are 62 and 8 microM, respectively. ADP is a competitive inhibitor with respect to ATP with an inhibition constant of 0.6 mM. All other product inhibition patterns including ADP versus fructose-6-P and fructose-2,6-P2 versus ATP and fructose-6-P are noncompetitive. Fructose-6-P,2-kinase also catalyzes the hydrolysis of ATP at about 10% of the rate of the overall reaction. There is no detectable burst of ADP formation upon incubating ATP with a stoichiometric amount of the enzyme. Similarly no burst of fructose-6-P formation is observed by reacting fructose-2,6-P2 with fructose 2,6-bisphosphatase. These results demonstrate that (a) the fructose-6-P,2-kinase reaction is consistent with a sequential mode of substrate addition rather than a "ping-pong" mechanism, (b) phosphorylenzyme , if formed, is not likely a kinetically important intermediate, and (c) no common phosphorylenzyme intermedite between fructose-6-P,2-kinase and fructose 2,6-bisphosphatase seems to exist.
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010770:Phosphotransferases / Q000378:metabolism*; D013053:Spectrophotometry
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases; D008274:Magnesium