Abstract
Liver and skeletal muscle isoforms of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF2K/Fru-2,6-P2ase) isoenzymes are products of alternatively spliced first exons of the same gene, with common kinase and bisphosphatase domains. The muscle-specific exon-1 encodes nine unique amino acids, that lack the cAMP-dependent protein kinase (PK-A) phosphorylation site, and differ in sequence from those encoded by the liver-specific exon-1 (32 amino acids), contributing to its much lower affinity for fructose 6-phosphate (Fru-6-P). PK-A phosphorylation of the liver isoform at Ser32 reduces the affinity of the kinase for Fru-6-P, and stimulates the bisphosphatase V(max). In the present study, we have defined the locus of interaction of the N-terminal residues with the N-terminal kinase and C-terminal domains by successive N- and C-terminal deletions. This study shows that: (1) residues Gly5-Glu6-Leu7 of the liver isoform are responsible for increasing the affinity of 6PF2K for Fru-6-P, maintaining the inhibition of Fru-2,6-P2ase activity, and mediating the effects of PK-A phosphorylation on the two activities; (2) the loss of Fru-6-P inhibition of the bisphosphatase and the enhancement of its V(max), rather than the inhibition of the kinase, may be responsible for the behaviour of the muscle isoform primarily as a bisphosphatase; (3) the composition of residues 24-32 of the liver form appears to confer the enhanced kinase catalytic rate of this form over that of the muscle isoform. It is concluded that specific regions of the N-terminus of liver and skeletal muscle 6PF2K/Fru-2,6-P2ase have a role in adapting the two activities to work in the physiological range of pH and substrate concentrations found in each particular tissue.
Original language | English (US) |
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Pages (from-to) | 459-467 |
Number of pages | 9 |
Journal | Biochemical Journal |
Volume | 347 |
Issue number | 2 |
DOIs | |
State | Published - Apr 15 2000 |
Externally published | Yes |
Keywords
- Enzyme kinetics
- Gluconeogenesis
- Glycolysis
- Hepatic glucose metabolism
- Structure/function
ASJC Scopus subject areas
- Biochemistry
- Molecular Biology
- Cell Biology