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 Vmax. 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 Vmax, 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.
N- and C-termini modulate the effects of pH and phosphorylation on hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase
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Irwin J. KURLAND, Brett CHAPMAN, M. Raafat EL-MAGHRABI; N- and C-termini modulate the effects of pH and phosphorylation on hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Biochem J 15 April 2000; 347 (2): 459–467. doi: https://doi.org/10.1042/bj3470459
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