Evidence for NH2- and COOH-terminal interactions in rat 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

Kai Lin, Irwin J. Kurland, Lin Li, Yong Hwan Lee, David Okar, James F. Marecek, Simon J. Pilkis

Research output: Contribution to journalArticle

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Abstract

The pH kinetic behavior of several rat fructose-2,6-bisphosphatase forms was analyzed. The bisphosphatase maximal velocity of the hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was optimal at pH 5, but decreased to 12% of the optimal value in the pH range 7.0-7.5. This decrease depended on deprotonation of a group with a pK of 5.7. In contrast, the separate bisphosphatase domain, a 30-amino acid COOH-terminal truncated form (CT30) of the liver enzyme, and the skeletal muscle bifunctional enzyme exhibited pH-insensitive maximal velocities which were 5-10-fold higher than that of the bisphosphatase of the liver bifunctional enzyme at pH 7.0-7.5. The pK values of the C-2 and C-6 phosphoryl groups were 6.0 and 5.75, respectively, as determined by 31P NMR. Analysis of log kcat/Km versus pH profiles revealed two pK values, one at 6.1, which probably is a substrate pK, and the other at 8.4, which represents an enzyme group. Protein kinasecatalyzed phosphorylation of the liver isoform activated the bisphosphatase, and the pK of the group seen in the kcat profile was increased from 5.7 to 6.4. However, phosphorylation of the CT30 mutant had no effect on the bisphosphatase. The data indicate that NH2- and COOH-terminal interactions in the liver bifunctional enzyme affect the pH dependence of the fructose-2,6-bisphosphatase and its activation by phosphorylation.

Original languageEnglish (US)
Pages (from-to)16953-16960
Number of pages8
JournalJournal of Biological Chemistry
Volume269
Issue number24
StatePublished - Jun 17 1994
Externally publishedYes

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Phosphofructokinase-2
Rats
Liver
Phosphorylation
Enzymes
Deprotonation
Muscle
Protein Isoforms
Chemical activation
Nuclear magnetic resonance
Amino Acids
Kinetics
Skeletal Muscle
Substrates
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Lin, K., Kurland, I. J., Li, L., Lee, Y. H., Okar, D., Marecek, J. F., & Pilkis, S. J. (1994). Evidence for NH2- and COOH-terminal interactions in rat 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Journal of Biological Chemistry, 269(24), 16953-16960.

Evidence for NH2- and COOH-terminal interactions in rat 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. / Lin, Kai; Kurland, Irwin J.; Li, Lin; Lee, Yong Hwan; Okar, David; Marecek, James F.; Pilkis, Simon J.

In: Journal of Biological Chemistry, Vol. 269, No. 24, 17.06.1994, p. 16953-16960.

Research output: Contribution to journalArticle

Lin, K, Kurland, IJ, Li, L, Lee, YH, Okar, D, Marecek, JF & Pilkis, SJ 1994, 'Evidence for NH2- and COOH-terminal interactions in rat 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase', Journal of Biological Chemistry, vol. 269, no. 24, pp. 16953-16960.
Lin, Kai ; Kurland, Irwin J. ; Li, Lin ; Lee, Yong Hwan ; Okar, David ; Marecek, James F. ; Pilkis, Simon J. / Evidence for NH2- and COOH-terminal interactions in rat 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. In: Journal of Biological Chemistry. 1994 ; Vol. 269, No. 24. pp. 16953-16960.
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abstract = "The pH kinetic behavior of several rat fructose-2,6-bisphosphatase forms was analyzed. The bisphosphatase maximal velocity of the hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was optimal at pH 5, but decreased to 12{\%} of the optimal value in the pH range 7.0-7.5. This decrease depended on deprotonation of a group with a pK of 5.7. In contrast, the separate bisphosphatase domain, a 30-amino acid COOH-terminal truncated form (CT30) of the liver enzyme, and the skeletal muscle bifunctional enzyme exhibited pH-insensitive maximal velocities which were 5-10-fold higher than that of the bisphosphatase of the liver bifunctional enzyme at pH 7.0-7.5. The pK values of the C-2 and C-6 phosphoryl groups were 6.0 and 5.75, respectively, as determined by 31P NMR. Analysis of log kcat/Km versus pH profiles revealed two pK values, one at 6.1, which probably is a substrate pK, and the other at 8.4, which represents an enzyme group. Protein kinasecatalyzed phosphorylation of the liver isoform activated the bisphosphatase, and the pK of the group seen in the kcat profile was increased from 5.7 to 6.4. However, phosphorylation of the CT30 mutant had no effect on the bisphosphatase. The data indicate that NH2- and COOH-terminal interactions in the liver bifunctional enzyme affect the pH dependence of the fructose-2,6-bisphosphatase and its activation by phosphorylation.",
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AU - Pilkis, Simon J.

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