Kinetic studies of fructose 6-phosphate,2-kinase and fructose 2,6-bisphosphatase

S. Kitajima, R. Sakakibara, K. Uyeda

Research output: Contribution to journalArticle

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Abstract

The reactions catalyzed by a bifunctional enzyme, fructose-6-P,2-kinase and fructose 2,6-bisphosphatase, 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 K(m) values for ATP and ADP for the reaction are 120 and 66 μM, 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 μM (1.6 x K(m)) 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. 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 μM, respectively, while those values for ADP and fructose-2,6-P2 are 62 and 8 μM, 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 intermediate between fructose-6-P,2-kinase and fructose 2,6-bisphosphatase seems to exist.

Original languageEnglish (US)
Pages (from-to)6896-6903
Number of pages8
JournalJournal of Biological Chemistry
Volume259
Issue number11
StatePublished - 1984

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Phosphofructokinase-2
Fructose
Kinetics
Adenosine Diphosphate
Adenosine Triphosphate
Isotopes
Substrates
Adenine Nucleotides
Enzymes
Hydrolysis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Kitajima, S., Sakakibara, R., & Uyeda, K. (1984). Kinetic studies of fructose 6-phosphate,2-kinase and fructose 2,6-bisphosphatase. Journal of Biological Chemistry, 259(11), 6896-6903.

Kinetic studies of fructose 6-phosphate,2-kinase and fructose 2,6-bisphosphatase. / Kitajima, S.; Sakakibara, R.; Uyeda, K.

In: Journal of Biological Chemistry, Vol. 259, No. 11, 1984, p. 6896-6903.

Research output: Contribution to journalArticle

Kitajima, S, Sakakibara, R & Uyeda, K 1984, 'Kinetic studies of fructose 6-phosphate,2-kinase and fructose 2,6-bisphosphatase', Journal of Biological Chemistry, vol. 259, no. 11, pp. 6896-6903.
Kitajima, S. ; Sakakibara, R. ; Uyeda, K. / Kinetic studies of fructose 6-phosphate,2-kinase and fructose 2,6-bisphosphatase. In: Journal of Biological Chemistry. 1984 ; Vol. 259, No. 11. pp. 6896-6903.
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abstract = "The reactions catalyzed by a bifunctional enzyme, fructose-6-P,2-kinase and fructose 2,6-bisphosphatase, 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 K(m) values for ATP and ADP for the reaction are 120 and 66 μM, 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 μM (1.6 x K(m)) 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. 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 μM, respectively, while those values for ADP and fructose-2,6-P2 are 62 and 8 μM, 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 intermediate between fructose-6-P,2-kinase and fructose 2,6-bisphosphatase seems to exist.",
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