Kinetic properties of bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from spinach leaves

Jonathan E. Markham, Nicholas J. Kruger

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

A cDNA encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was isolated from a Spinacia oleracea leaf library and used to express a recombinant enzyme in Escherichia coli and Spodoptera frugiperda cells. The insoluble protein expressed in E. coli was purified and used to raise antibodies. Western blot analysis of a protein extract from spinach leaf showed a single band of 90.8 kDa. Soluble protein was purified to homogeneity from S. frugiperda cells infected with recombinant baculovirus harboring the isolated cDNA. The soluble protein had a molecular mass of 320 kDa, estimated by gel filtration chromatography, and a subunit size of 90.8 kDa. The purified protein had activity of both 6-phosphofructo-2-kinase (specific activity 10.4-15.9 nmolmin 1mg protein 1) and fructose-2,6-bisphosphatase (specific activity 1.65-1.75 nmolmin 1mg protein 1). The 6-phosphofructo-2-kinase activity was activated by inorganic phosphate, and inhibited by 3-carbon phosphorylated metabolites and pyrophosphate. In the presence of phosphate, 3-phosphoglycerate was a mixed inhibitor with respect to both fructose 6-phosphate and ATP. Fructose-2,6-bisphosphatase activity was sensitive to product inhibition; inhibition by inorganic phosphate was uncompetitive, whereas inhibition by fructose 6-phosphate was mixed. These kinetic properties support the view that the level of fructose 2,6-bisphosphate in leaves is determined by the relative concentrations of hexose phosphates, three-carbon phosphate esters and inorganic phosphate in the cytosol through reciprocal modulation of 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase activities of the bifunctional enzyme.

Original languageEnglish (US)
Pages (from-to)1267-1277
Number of pages11
JournalEuropean Journal of Biochemistry
Volume269
Issue number4
DOIs
StatePublished - Mar 7 2002

Fingerprint

Phosphofructokinase-2
Spinacia oleracea
Phosphates
Kinetics
Proteins
Spodoptera
Escherichia coli
Carbon
Complementary DNA
Hexoses
Baculoviridae
Molecular mass
Enzymes
Metabolites
Chromatography
Cytosol
Libraries
Gel Chromatography
Esters
Adenosine Triphosphate

Keywords

  • 6-Phosphofructo-2-kinase
  • Fructose 2,6-bisphosphate
  • Fructose-2,6-bisphosphatase
  • Spinach leaf
  • Spinacia oleracea

ASJC Scopus subject areas

  • Biochemistry

Cite this

Kinetic properties of bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from spinach leaves. / Markham, Jonathan E.; Kruger, Nicholas J.

In: European Journal of Biochemistry, Vol. 269, No. 4, 07.03.2002, p. 1267-1277.

Research output: Contribution to journalArticle

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abstract = "A cDNA encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was isolated from a Spinacia oleracea leaf library and used to express a recombinant enzyme in Escherichia coli and Spodoptera frugiperda cells. The insoluble protein expressed in E. coli was purified and used to raise antibodies. Western blot analysis of a protein extract from spinach leaf showed a single band of 90.8 kDa. Soluble protein was purified to homogeneity from S. frugiperda cells infected with recombinant baculovirus harboring the isolated cDNA. The soluble protein had a molecular mass of 320 kDa, estimated by gel filtration chromatography, and a subunit size of 90.8 kDa. The purified protein had activity of both 6-phosphofructo-2-kinase (specific activity 10.4-15.9 nmolmin 1mg protein 1) and fructose-2,6-bisphosphatase (specific activity 1.65-1.75 nmolmin 1mg protein 1). The 6-phosphofructo-2-kinase activity was activated by inorganic phosphate, and inhibited by 3-carbon phosphorylated metabolites and pyrophosphate. In the presence of phosphate, 3-phosphoglycerate was a mixed inhibitor with respect to both fructose 6-phosphate and ATP. Fructose-2,6-bisphosphatase activity was sensitive to product inhibition; inhibition by inorganic phosphate was uncompetitive, whereas inhibition by fructose 6-phosphate was mixed. These kinetic properties support the view that the level of fructose 2,6-bisphosphate in leaves is determined by the relative concentrations of hexose phosphates, three-carbon phosphate esters and inorganic phosphate in the cytosol through reciprocal modulation of 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase activities of the bifunctional enzyme.",
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