Effect of adding phosphorylation sites for cAMP-dependent protein kinase to rat testis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

Yumiko Abe, Kosaku Uyeda

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9 Citations (Scopus)

Abstract

In contrast to liver and heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases, the testis isozyme lacks a phosphorylation site for cAMP-dependent protein kinase. In order to determine the effect of phosphorylation site location for the protein kinase on rat testis bifunctional enzyme, consensus amino acid sequences (RRXS) were added at different distances from the N-terminus by site-directed mutagenesis. The expressed wild-type enzyme (WT) and mutant enzymes containing a phosphorylation site at Ser7 (mutant enzyme RT2KS7, where RT2K = rat testis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase), Ser15 (RT2KS15), or Ser30 (RT2KS30) were purified to apparent homogeneity. All the mutant enzymes served as substrates for the protein kinase, and the phosphate incorporation was over 90%. The Km values of protein kinase A for RT2KS7, RT2KS15, and RT2KS30 were 250 μM, 110 μM, and 50 μM, respectively, and the relative rates were 1, 8, and 23. Various kinetic parameters of dephospho and phospho forms of these enzymes were determined. The kinetic constants of the dephospho form of RT2KS30 were similar to those of WT, but those of RT2KS15 and RT2KS7 showed an 8-fold increase in Km Fru6P, an approximately 30% decrease in the Fru-6-P,2-kinase activity, and a 3-fold increase in fructose-2,6-bisphosphatase activity. Phosphorylation of RT2KS30 resulted in a shift in the Fru-6-P saturation curve from Michaelis-Menten kinetics to sigmoidal, with increased Km Fru6P and activation of fructose-2,6-bisphosphatase. The kinetic constants of RT2KS15 and RT2KS7 were not altered by phosphorylation. All the mutant enzymes were more sensitive to heat inactivation than was WT. Furthermore, the phospho-RT2KS30 was more thermally labile than its dephospho form. A Stern-Volmer plot of iodide quenching of RT2KS30 tryptophan fluorescence was nonlinear, but those of the other mutant enzymes were linear. These results suggest that all tryptophans in the RT2KS15 and RT2KPS7 mutant enzymes are more exposed and accessible to iodide than RT2KS30.

Original languageEnglish (US)
Pages (from-to)5766-5771
Number of pages6
JournalBiochemistry®
Volume33
Issue number19
StatePublished - 1994

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Phosphofructokinase-2
Phosphorylation
Cyclic AMP-Dependent Protein Kinases
Testis
Rats
Enzymes
Iodides
Tryptophan
Protein Kinases
Kinetics
Mutagenesis
Site-Directed Mutagenesis
Fructose
Kinetic parameters
Liver
Isoenzymes
Amino Acid Sequence
Quenching

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{dc2b91c8df84477cbe743f26266e33fa,
title = "Effect of adding phosphorylation sites for cAMP-dependent protein kinase to rat testis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase",
abstract = "In contrast to liver and heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases, the testis isozyme lacks a phosphorylation site for cAMP-dependent protein kinase. In order to determine the effect of phosphorylation site location for the protein kinase on rat testis bifunctional enzyme, consensus amino acid sequences (RRXS) were added at different distances from the N-terminus by site-directed mutagenesis. The expressed wild-type enzyme (WT) and mutant enzymes containing a phosphorylation site at Ser7 (mutant enzyme RT2KS7, where RT2K = rat testis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase), Ser15 (RT2KS15), or Ser30 (RT2KS30) were purified to apparent homogeneity. All the mutant enzymes served as substrates for the protein kinase, and the phosphate incorporation was over 90{\%}. The Km values of protein kinase A for RT2KS7, RT2KS15, and RT2KS30 were 250 μM, 110 μM, and 50 μM, respectively, and the relative rates were 1, 8, and 23. Various kinetic parameters of dephospho and phospho forms of these enzymes were determined. The kinetic constants of the dephospho form of RT2KS30 were similar to those of WT, but those of RT2KS15 and RT2KS7 showed an 8-fold increase in Km Fru6P, an approximately 30{\%} decrease in the Fru-6-P,2-kinase activity, and a 3-fold increase in fructose-2,6-bisphosphatase activity. Phosphorylation of RT2KS30 resulted in a shift in the Fru-6-P saturation curve from Michaelis-Menten kinetics to sigmoidal, with increased Km Fru6P and activation of fructose-2,6-bisphosphatase. The kinetic constants of RT2KS15 and RT2KS7 were not altered by phosphorylation. All the mutant enzymes were more sensitive to heat inactivation than was WT. Furthermore, the phospho-RT2KS30 was more thermally labile than its dephospho form. A Stern-Volmer plot of iodide quenching of RT2KS30 tryptophan fluorescence was nonlinear, but those of the other mutant enzymes were linear. These results suggest that all tryptophans in the RT2KS15 and RT2KPS7 mutant enzymes are more exposed and accessible to iodide than RT2KS30.",
author = "Yumiko Abe and Kosaku Uyeda",
year = "1994",
language = "English (US)",
volume = "33",
pages = "5766--5771",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "19",

}

TY - JOUR

T1 - Effect of adding phosphorylation sites for cAMP-dependent protein kinase to rat testis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

AU - Abe, Yumiko

AU - Uyeda, Kosaku

PY - 1994

Y1 - 1994

N2 - In contrast to liver and heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases, the testis isozyme lacks a phosphorylation site for cAMP-dependent protein kinase. In order to determine the effect of phosphorylation site location for the protein kinase on rat testis bifunctional enzyme, consensus amino acid sequences (RRXS) were added at different distances from the N-terminus by site-directed mutagenesis. The expressed wild-type enzyme (WT) and mutant enzymes containing a phosphorylation site at Ser7 (mutant enzyme RT2KS7, where RT2K = rat testis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase), Ser15 (RT2KS15), or Ser30 (RT2KS30) were purified to apparent homogeneity. All the mutant enzymes served as substrates for the protein kinase, and the phosphate incorporation was over 90%. The Km values of protein kinase A for RT2KS7, RT2KS15, and RT2KS30 were 250 μM, 110 μM, and 50 μM, respectively, and the relative rates were 1, 8, and 23. Various kinetic parameters of dephospho and phospho forms of these enzymes were determined. The kinetic constants of the dephospho form of RT2KS30 were similar to those of WT, but those of RT2KS15 and RT2KS7 showed an 8-fold increase in Km Fru6P, an approximately 30% decrease in the Fru-6-P,2-kinase activity, and a 3-fold increase in fructose-2,6-bisphosphatase activity. Phosphorylation of RT2KS30 resulted in a shift in the Fru-6-P saturation curve from Michaelis-Menten kinetics to sigmoidal, with increased Km Fru6P and activation of fructose-2,6-bisphosphatase. The kinetic constants of RT2KS15 and RT2KS7 were not altered by phosphorylation. All the mutant enzymes were more sensitive to heat inactivation than was WT. Furthermore, the phospho-RT2KS30 was more thermally labile than its dephospho form. A Stern-Volmer plot of iodide quenching of RT2KS30 tryptophan fluorescence was nonlinear, but those of the other mutant enzymes were linear. These results suggest that all tryptophans in the RT2KS15 and RT2KPS7 mutant enzymes are more exposed and accessible to iodide than RT2KS30.

AB - In contrast to liver and heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases, the testis isozyme lacks a phosphorylation site for cAMP-dependent protein kinase. In order to determine the effect of phosphorylation site location for the protein kinase on rat testis bifunctional enzyme, consensus amino acid sequences (RRXS) were added at different distances from the N-terminus by site-directed mutagenesis. The expressed wild-type enzyme (WT) and mutant enzymes containing a phosphorylation site at Ser7 (mutant enzyme RT2KS7, where RT2K = rat testis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase), Ser15 (RT2KS15), or Ser30 (RT2KS30) were purified to apparent homogeneity. All the mutant enzymes served as substrates for the protein kinase, and the phosphate incorporation was over 90%. The Km values of protein kinase A for RT2KS7, RT2KS15, and RT2KS30 were 250 μM, 110 μM, and 50 μM, respectively, and the relative rates were 1, 8, and 23. Various kinetic parameters of dephospho and phospho forms of these enzymes were determined. The kinetic constants of the dephospho form of RT2KS30 were similar to those of WT, but those of RT2KS15 and RT2KS7 showed an 8-fold increase in Km Fru6P, an approximately 30% decrease in the Fru-6-P,2-kinase activity, and a 3-fold increase in fructose-2,6-bisphosphatase activity. Phosphorylation of RT2KS30 resulted in a shift in the Fru-6-P saturation curve from Michaelis-Menten kinetics to sigmoidal, with increased Km Fru6P and activation of fructose-2,6-bisphosphatase. The kinetic constants of RT2KS15 and RT2KS7 were not altered by phosphorylation. All the mutant enzymes were more sensitive to heat inactivation than was WT. Furthermore, the phospho-RT2KS30 was more thermally labile than its dephospho form. A Stern-Volmer plot of iodide quenching of RT2KS30 tryptophan fluorescence was nonlinear, but those of the other mutant enzymes were linear. These results suggest that all tryptophans in the RT2KS15 and RT2KPS7 mutant enzymes are more exposed and accessible to iodide than RT2KS30.

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M3 - Article

VL - 33

SP - 5766

EP - 5771

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

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