Successive deletion of up lo 22 ainino arids from the NH-lenmnu;- of rat hepatic PF2K-B was done in order to investigate the mechanism by which NH2/OOOH interactions are involved in (he regulation of ! he enzyme liv cAMP-dependent phosphorylation. Deletion of the first 3 amino arid-; did not affect kinase or hisphosphata.se activities. Deletion of the first 6, 11.or 22 residues caused a progressive decrease in the kinase Vmax. and iarrensc in its SO.5 for Fru 6-P, and an increa.se in the bibphosphata.se Vrnax. PhosplHnv lalion induced reciprocal changes in the enzyme's artivitie-. were diminished, but not abolished. A JO-fold increase in the SO.5 for Fra-6-P and a 2 fold increase in the bisphosphatase Vmax occurred when the first fi residues where removed. A >95% rediu'tion in 6PF2K Vmax. was observed in the (llufi to Ala mutated enzyme. The Vmax of the liver isofonn showed a bimodai pH dependence, that became unimodal by deletion of fi or more NH2-1ermin;d residues. It is concluded that : 1 ) the NH2-torminal residues Met4-Gly-Glu6 of hepatic PF2K-B interacts with the kinase active site to increase ils Vmax and affinity for Fru 6-P. as weli as to facilitate phosphorylation induced acliutv changes, with Glut} playing a, particularly important role in this interact inn; 2) the initial portion of the NH2-terrninus is important in adapting 6PF 2-K kinetics to the intracelluiar pII range.
|Original language||English (US)|
|State||Published - Dec 1 1997|
ASJC Scopus subject areas
- Molecular Biology