The kinase activity of human brain 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is regulated via inhibition by phosphoenolpyruvate.
The two enzymatic activities of the highly conserved catalytic core of 6PF2K/Fru-2,6-P(2)ase are thought to be reciprocally regulated by the amino- and carboxy-terminal regions unique to each isoform. In this study, we describe the recombinant expression, purification, and kinetic characterization of two human brain 6PF2K/Fru-2,6-P(2)ase splice variants, HBP1 and HBP2. Interestingly, both lack an arginine which is highly conserved among other tissue isoforms, and which is understood to be critical to the fructose-2,6-bisphosphatase mechanism. As a result, the phosphatase activity of both HBP isoforms is negligible, but we found that it could be recovered by restoration of the arginine by site directed mutagenesis. We also found that AMP activated protein kinase and protein kinases A, B, and C catalyzed the phosphorylation of Ser-460 of HBP1, and that in addition both isoforms are phosphorylated at a second, as yet undetermined site by protein kinase C. However, none of the phosphorylations had any effect on the intrinsic kinetic characteristics of either enzymatic activity, and neither did point mutation (mimicking phosphorylation), deletion, and alternative-splice modification of the HBP1 carboxy-terminal region. Instead, these phosphorylations and mutations decreased the sensitivity of the 6PF2K to a potent allosteric inhibitor, phosphoenolpyruvate, which appears to be the major regulatory mechanism.
Manes
Nathan P
NP
Department of Physiology and Biophysics, Stony Brook University, NY 11794-8661, USA.
El-Maghrabi
M Raafat
MR
D055372:AMP-Activated Protein Kinases; D000495:Allosteric Site; D017398:Alternative Splicing; D001120:Arginine / Q000737:chemistry; D001921:Brain / Q000201:enzymology* / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic; D015723:Gene Library; D006801:Humans; D007700:Kinetics; D015233:Models, Statistical; D009097:Multienzyme Complexes / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010728:Phosphoenolpyruvate / Q000378:metabolism*; D025481:Phosphofructokinase-2 / Q000096:biosynthesis* / Q000737:chemistry*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D011994:Recombinant Proteins / Q000737:chemistry; D012694:Serine / Q000737:chemistry
D009097:Multienzyme Complexes; D020033:Protein Isoforms; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D012694:Serine; D010728:Phosphoenolpyruvate; D004247:DNA; D001120:Arginine; D025481:Phosphofructokinase-2; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D011493:Protein Kinase C; D055372:AMP-Activated Protein Kinases; D010744:Phosphoric Monoester Hydrolases