N- and C-termini modulate the effects of pH and phosphorylation on hepatic6-phosphofructo-2-kinase/fructose-2,6-biphosphatase

Liver and skeletal muscle isoforms of 6-phosphofructo-2-kinase/fructose-2,6 -bisphosphatase (6PF2K/Fru-2,6-P(2)ase) isoenzymes are products of alternat ively spliced first exons of the same gene, with common kinase and bisphosp hatase domains. The muscle-specific exon-1 encodes nine unique amino acids, that lack the cAMP-dependent protein kinase (PK-A) phosphorylation site, a nd differ in sequence from those encoded by the liver-specific exon-1 (32 a mino acids), contributing to its much lower affinity for fructose 6-phospha te (Fru-6-P). PK-A phosphorylation of the liver isoform at Ser(32) 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 s uccessive N- and C-terminal deletions. This study shows that: (1) residues Gly(5)-Glu(6)-Leu(7) of the liver isoform are responsible for increasing th e affinity of 6PF2K for Fru-6-P, maintaining the inhibition of Fru-2,6-P(2) ase 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 th e enhancement of its V-max, rather than the inhibition of the kinase, may b e responsible for the behaviour of the muscle isoform primarily as a bispho sphatase; (3) the composition of residues 24-32 of the liver form appears t o confer the enhanced kinase catalytic rate of this form over that of the m uscle isoform. It is concluded that specific regions of the N-terminus of l iver and skeletal muscle 6PF2K/Fru-2,6-P(2)ase have a role in adapting the two activities to work in the physiological range of pH and substrate conce ntrations found in each particular tissue.