Expression of bovine heart fructose 6-phosphate,2-kinase: fructose 2,6- bisphosphatase and determination of the role of the carboxyl terminus by mutagenesis

Y. Abe, Y. Minami, Y. Li, C. Nguyen, K. Uyeda

Research output: Contribution to journalArticle

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Abstract

Bovine heart fructose 6-P,2-kinase:fructose 2,6-bisphosphatase was expressed in Escherichia coli. In order to determine the role of the carboxyl-terminal peptide, 49 and 78 amino acids from the C-terminus were deleted using oligonucleotide-directed mutagenesis. The expressed wild-type and mutant enzymes were purified to homogeneity, and the steady-state kinetics of the mutant enzymes were compared to those of the wild-type enzyme. Deletion of 49 residues (Del 49) resulted in a 35% decrease in Km(Fru6P), a 36% increase in V(max), and a 2-fold increase in k(cat)/K(m) of the kinase. There was no change in the kinetic properties of the phosphatase activity. Deletion of 78 residues (Del 78) resulted in a 4.5-fold decrease in K(m)(Fru6P), a 2.5-fold increase in V(max), a 12-fold increase in k(cat)/K(m) of the kinase, and a 3-fold increase in k(cat)/K(m) of the phosphatase. Phosphorylation of the wild-type and Del 49 enzymes resulted in decreased K(m)(Fru6P) and activation of the kinase without affecting the phosphatase activity. Thermal inactivation rates of the wild-type and Del 49 enzymes were similar, but the rate of Del 78 was more rapid. The phosphorylated wild-type and Del 49 enzymes were more sensitive to thermal inactivation than the dephospho forms. Urea inactivation of the kinase and phosphatase of wild- type and Del 49 were similar, but Del 78 was more sensitive to urea. All phosphorylated enzymes were more susceptible to urea inactivation. These results suggest that the C-terminal peptide of the enzyme, especially the region Phe453-Asn482, containing protein kinase A and C phosphorylation sites, is important in maintaining less active (T) states of the kinase and the phosphatase domains. Phosphorylation of the peptide converts the kinase to a more active (R) state without affecting the phosphatase, but deletion of the peptide results in activation of the phosphatase to R state.

Original languageEnglish (US)
Pages (from-to)2553-2559
Number of pages7
JournalBiochemistry
Volume34
Issue number8
DOIs
StatePublished - 1995

Fingerprint

Phosphofructokinase-2
Mutagenesis
Phosphoric Monoester Hydrolases
Phosphotransferases
Enzymes
Phosphorylation
Urea
Peptides
Hot Temperature
Chemical activation
Kinetics
Site-Directed Mutagenesis
Cyclic AMP-Dependent Protein Kinases
Oligonucleotides
Escherichia coli
Protein Kinase C
Amino Acids

ASJC Scopus subject areas

  • Biochemistry

Cite this

Expression of bovine heart fructose 6-phosphate,2-kinase : fructose 2,6- bisphosphatase and determination of the role of the carboxyl terminus by mutagenesis. / Abe, Y.; Minami, Y.; Li, Y.; Nguyen, C.; Uyeda, K.

In: Biochemistry, Vol. 34, No. 8, 1995, p. 2553-2559.

Research output: Contribution to journalArticle

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abstract = "Bovine heart fructose 6-P,2-kinase:fructose 2,6-bisphosphatase was expressed in Escherichia coli. In order to determine the role of the carboxyl-terminal peptide, 49 and 78 amino acids from the C-terminus were deleted using oligonucleotide-directed mutagenesis. The expressed wild-type and mutant enzymes were purified to homogeneity, and the steady-state kinetics of the mutant enzymes were compared to those of the wild-type enzyme. Deletion of 49 residues (Del 49) resulted in a 35{\%} decrease in Km(Fru6P), a 36{\%} increase in V(max), and a 2-fold increase in k(cat)/K(m) of the kinase. There was no change in the kinetic properties of the phosphatase activity. Deletion of 78 residues (Del 78) resulted in a 4.5-fold decrease in K(m)(Fru6P), a 2.5-fold increase in V(max), a 12-fold increase in k(cat)/K(m) of the kinase, and a 3-fold increase in k(cat)/K(m) of the phosphatase. Phosphorylation of the wild-type and Del 49 enzymes resulted in decreased K(m)(Fru6P) and activation of the kinase without affecting the phosphatase activity. Thermal inactivation rates of the wild-type and Del 49 enzymes were similar, but the rate of Del 78 was more rapid. The phosphorylated wild-type and Del 49 enzymes were more sensitive to thermal inactivation than the dephospho forms. Urea inactivation of the kinase and phosphatase of wild- type and Del 49 were similar, but Del 78 was more sensitive to urea. All phosphorylated enzymes were more susceptible to urea inactivation. These results suggest that the C-terminal peptide of the enzyme, especially the region Phe453-Asn482, containing protein kinase A and C phosphorylation sites, is important in maintaining less active (T) states of the kinase and the phosphatase domains. Phosphorylation of the peptide converts the kinase to a more active (R) state without affecting the phosphatase, but deletion of the peptide results in activation of the phosphatase to R state.",
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T1 - Expression of bovine heart fructose 6-phosphate,2-kinase

T2 - fructose 2,6- bisphosphatase and determination of the role of the carboxyl terminus by mutagenesis

AU - Abe, Y.

AU - Minami, Y.

AU - Li, Y.

AU - Nguyen, C.

AU - Uyeda, K.

PY - 1995

Y1 - 1995

N2 - Bovine heart fructose 6-P,2-kinase:fructose 2,6-bisphosphatase was expressed in Escherichia coli. In order to determine the role of the carboxyl-terminal peptide, 49 and 78 amino acids from the C-terminus were deleted using oligonucleotide-directed mutagenesis. The expressed wild-type and mutant enzymes were purified to homogeneity, and the steady-state kinetics of the mutant enzymes were compared to those of the wild-type enzyme. Deletion of 49 residues (Del 49) resulted in a 35% decrease in Km(Fru6P), a 36% increase in V(max), and a 2-fold increase in k(cat)/K(m) of the kinase. There was no change in the kinetic properties of the phosphatase activity. Deletion of 78 residues (Del 78) resulted in a 4.5-fold decrease in K(m)(Fru6P), a 2.5-fold increase in V(max), a 12-fold increase in k(cat)/K(m) of the kinase, and a 3-fold increase in k(cat)/K(m) of the phosphatase. Phosphorylation of the wild-type and Del 49 enzymes resulted in decreased K(m)(Fru6P) and activation of the kinase without affecting the phosphatase activity. Thermal inactivation rates of the wild-type and Del 49 enzymes were similar, but the rate of Del 78 was more rapid. The phosphorylated wild-type and Del 49 enzymes were more sensitive to thermal inactivation than the dephospho forms. Urea inactivation of the kinase and phosphatase of wild- type and Del 49 were similar, but Del 78 was more sensitive to urea. All phosphorylated enzymes were more susceptible to urea inactivation. These results suggest that the C-terminal peptide of the enzyme, especially the region Phe453-Asn482, containing protein kinase A and C phosphorylation sites, is important in maintaining less active (T) states of the kinase and the phosphatase domains. Phosphorylation of the peptide converts the kinase to a more active (R) state without affecting the phosphatase, but deletion of the peptide results in activation of the phosphatase to R state.

AB - Bovine heart fructose 6-P,2-kinase:fructose 2,6-bisphosphatase was expressed in Escherichia coli. In order to determine the role of the carboxyl-terminal peptide, 49 and 78 amino acids from the C-terminus were deleted using oligonucleotide-directed mutagenesis. The expressed wild-type and mutant enzymes were purified to homogeneity, and the steady-state kinetics of the mutant enzymes were compared to those of the wild-type enzyme. Deletion of 49 residues (Del 49) resulted in a 35% decrease in Km(Fru6P), a 36% increase in V(max), and a 2-fold increase in k(cat)/K(m) of the kinase. There was no change in the kinetic properties of the phosphatase activity. Deletion of 78 residues (Del 78) resulted in a 4.5-fold decrease in K(m)(Fru6P), a 2.5-fold increase in V(max), a 12-fold increase in k(cat)/K(m) of the kinase, and a 3-fold increase in k(cat)/K(m) of the phosphatase. Phosphorylation of the wild-type and Del 49 enzymes resulted in decreased K(m)(Fru6P) and activation of the kinase without affecting the phosphatase activity. Thermal inactivation rates of the wild-type and Del 49 enzymes were similar, but the rate of Del 78 was more rapid. The phosphorylated wild-type and Del 49 enzymes were more sensitive to thermal inactivation than the dephospho forms. Urea inactivation of the kinase and phosphatase of wild- type and Del 49 were similar, but Del 78 was more sensitive to urea. All phosphorylated enzymes were more susceptible to urea inactivation. These results suggest that the C-terminal peptide of the enzyme, especially the region Phe453-Asn482, containing protein kinase A and C phosphorylation sites, is important in maintaining less active (T) states of the kinase and the phosphatase domains. Phosphorylation of the peptide converts the kinase to a more active (R) state without affecting the phosphatase, but deletion of the peptide results in activation of the phosphatase to R state.

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