Significance of the amino terminus of rat testis fructose-6-phosphate,2-kinase: fructose-2,6-bisphosphatase

Nobuaki Tominaga, Yoshiko Minami, Ryuzo Sakakibara, Kosaku Uyeda

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Abstract

In order to assess the functional properties of the extreme amino-terminal peptide of rat testis fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase, 24 and 30 amino acids from the NH2 terminus were deleted using oligonucleotide-directed mutagenesis. The expressed wild-type (WT) and the mutant (Del 24 and Del 30) enzymes were purified to homogeneity. These enzymes were homodimers of subunit Mr values 54,000, 51,300, and 50,600. Deletion of 1-24 and 1-30 residues resulted in over 70% loss of Fru-6-P,2-kinase activity and 2-fold increase in Fru-2,6-bisphosphatase. Km Fru-6-P increased 7.5-fold, but there was no significant change in Km Fru-2,6P2 of the phosphatase. These mutant enzymes were more susceptible to protein concentration-dependent dissociation and thermal inactivation. In 2 M urea the kinase and the phosphatase activities of WT increased 17 and 56%, respectively, but no activation of any of the mutant enzymes was observed. At higher urea concentrations, all the enzymes were inactivated. Unfolding in urea, followed with intrinsic tryptophan fluorescence, showed that the unfolding proceeded in at least two stages. Fluorescence intensity at 338 nm of all these enzymes decreased below 2 M urea in which the enzyme activities remained the same (Del 24) or activated (WT). The second stage of unfolding was seen as the maximum shifted to 342 nm in 2-4 M urea, presumably involving dissociation. Complete unfolding above 4 M urea resulted in further shift in fluorescence maximum to 350 nm.

Original languageEnglish (US)
Pages (from-to)15951-15957
Number of pages7
JournalJournal of Biological Chemistry
Volume268
Issue number21
StatePublished - Jul 25 1993

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Phosphofructokinase-2
Urea
Testis
Rats
Enzymes
Fluorescence
Phosphoric Monoester Hydrolases
Mutagenesis
Enzyme activity
Oligonucleotides
Tryptophan
Site-Directed Mutagenesis
Phosphotransferases
Chemical activation
Hot Temperature
Amino Acids
Peptides

ASJC Scopus subject areas

  • Biochemistry

Cite this

Significance of the amino terminus of rat testis fructose-6-phosphate,2-kinase : fructose-2,6-bisphosphatase. / Tominaga, Nobuaki; Minami, Yoshiko; Sakakibara, Ryuzo; Uyeda, Kosaku.

In: Journal of Biological Chemistry, Vol. 268, No. 21, 25.07.1993, p. 15951-15957.

Research output: Contribution to journalArticle

Tominaga, Nobuaki ; Minami, Yoshiko ; Sakakibara, Ryuzo ; Uyeda, Kosaku. / Significance of the amino terminus of rat testis fructose-6-phosphate,2-kinase : fructose-2,6-bisphosphatase. In: Journal of Biological Chemistry. 1993 ; Vol. 268, No. 21. pp. 15951-15957.
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abstract = "In order to assess the functional properties of the extreme amino-terminal peptide of rat testis fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase, 24 and 30 amino acids from the NH2 terminus were deleted using oligonucleotide-directed mutagenesis. The expressed wild-type (WT) and the mutant (Del 24 and Del 30) enzymes were purified to homogeneity. These enzymes were homodimers of subunit Mr values 54,000, 51,300, and 50,600. Deletion of 1-24 and 1-30 residues resulted in over 70{\%} loss of Fru-6-P,2-kinase activity and 2-fold increase in Fru-2,6-bisphosphatase. Km Fru-6-P increased 7.5-fold, but there was no significant change in Km Fru-2,6P2 of the phosphatase. These mutant enzymes were more susceptible to protein concentration-dependent dissociation and thermal inactivation. In 2 M urea the kinase and the phosphatase activities of WT increased 17 and 56{\%}, respectively, but no activation of any of the mutant enzymes was observed. At higher urea concentrations, all the enzymes were inactivated. Unfolding in urea, followed with intrinsic tryptophan fluorescence, showed that the unfolding proceeded in at least two stages. Fluorescence intensity at 338 nm of all these enzymes decreased below 2 M urea in which the enzyme activities remained the same (Del 24) or activated (WT). The second stage of unfolding was seen as the maximum shifted to 342 nm in 2-4 M urea, presumably involving dissociation. Complete unfolding above 4 M urea resulted in further shift in fluorescence maximum to 350 nm.",
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