Bovine brain 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Evidence for a neural-specific isozyme (original) (raw)
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6-PHOSPHOFRUCTO-2-KINASE/FRUCTOSE-2,6-BISPHOSPHATASE in Rat Brain
The Biochemical journal, 1991
The concentration of fructose 2,6-bisphosphate in the brain remained stable during starvation and early stages of ischaemia, but decreased in diabetes or after lengthened ischaemia. 6-Phosphofructo-1-kinase activity was also decreased in diabetic and ischaemic animals, whereas 6-phosphofructo-2-kinase was not modified. The concentration of the bisphosphorylated metabolite seems to be remarkably constant under a wide variety of experimental conditions, suggesting that it plays an essential role in the basal activation of 6-phosphofructo-1-kinase. Purified 6-phosphofructo-2-kinase also showed fructose-2,6-bisphosphatase activity with an activity ratio similar to that of the purified heart isoenzyme. The brain enzyme also has a net charge similar to that of the heart isoenzyme. Its activity is not modified by sn-glycerol 3-phosphate, and it is more sensitive to citrate than the liver or muscle isoenzyme. Moreover, the enzyme from brain, similarly to that from heart and muscle, is not m...
Unequivocal demonstration of fructose-1,6-bisphosphatase in mammalian brain
Proceedings of the National Academy of Sciences, 1977
Fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase; EC 3.1.3.11) has been found in rat brain and identified unequivocally. The enzyme has been purified to 95% homogeneity by standard procedures, including adsorption to a phosphocellulose column followed by elution with substrate. The purified enzyme exhibits a broad optimum above pH 7.6. Both fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate are substrates of this enzyme; the hydrolysis of the latter occurs at about 20% of the rate of the former, and the Km for fructose 1,6-bisphosphate is approximately 1.32 X 10(-4) M. 5'-AMP, an inhibitor of other mammalian-fructose-1,6-bisphosphatases, is without effect, and in further contrast with the other enzymes there is no metal requirement for activity. Purified brain enzyme fails to crossreact with the antibody prepared against the purified liver fructose-1,6-bisphosphatase. On the other hand, antiserum produced against the brain fructose-1,6-bispho...
Archives of Biochemistry and Biophysics, 2005
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.
Proceedings of the National Academy of Sciences, 1990
We have shown previously that bovine heart fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase (EC 2.7.1.105/3.1.3.46) is phosphorylated by cAMPdependent protein kinase and protein kinase C; phosphorylation results in activation of kinase. This activation of heart enzyme is in contrast to results with the liver isozyme, in which phosphorylation by cAMP-dependent protein kinase inhibits the kinase activity. As an initial step toward understanding this difference between the isozymes we have determined the DNA sequence of the heart enzyme and analyzed the amino acid sequence with special emphasis on the location of the phosphorylation site. We isolated and sequenced two overlapping cDNA fragments, which together could encode the complete amino acid sequence of bovine heart fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase, a protein of 530 amino acids, with a calculated molecular weight of 60,679. Since the deduced protein contained amino acid sequences identical to the sequences of four known tryptic peptides from this enzyme we concluded that the deduced protein sequence did represent bovine heart enzyme. In addition, a cDNA fragment hybridized to a 4-kilobase mRNA from bovine heart. The phosphorylation sites of the heart enzyme were located near the C terminus, whereas the phosphorylation site of the liver isozyme is known to be located near the N terminus. These opposite locations of the phosphorylation sites may explain the contrasting effect of the covalent modification on the enzymes' activities.
Biochemistry, 1995
Bovine heart fructose 6-P,2-kinase:fructose 2,6-bisphosphatase was expressed in Escherichia coli. In order to determine the role of the carboxyl-terminal peptide, 49 and 78 amino acids from the C-terminus were deleted using oligonucleotide-directed mutagenesis. The expressed wild-type and mutant enzymes were purified to homogeneity, and the steady-state kinetics of the mutant enzymes were compared to those of the wild-type enzyme. Deletion of 49 residues (Del 49) resulted in a 35% decrease in KmFN6p, a 36% increase in Vm,, and a 2-fold increase in kcat/Km of the kinase. There was no change in the kinetic properties of the phosphatase activity. Deletion of 78 residues (Del 78) resulted in a 4.5-fold decrease in KmFN6P, a 2.5-fold increase in Vmm, a 12-fold increase in k&Km of the kinase, and a 3-fold increase in kcat/Km of the phosphatase. Phosphorylation of the wild-type and Del 49 enzymes resulted in decreased KmFN6' and activation of the kinase without affecting the phosphatase activity. Thermal inactivation rates of the wild-type and Del 49 enzymes were similar, but the rate of Del 78 was more rapid. The phosphorylated wild-type and Del 49 enzymes were more sensitive to thermal inactivation than the dephospho forms. Urea inactivation of the kinase and phosphatase of wild-type and Del 49 were similar, but Del 78 was more sensitive to urea. All phosphorylated enzymes were more susceptible to urea inactivation. These results suggest that the C-terminal peptide of the enzyme, especially the region Phe453containing protein kinase A and C phosphorylation sites, is important in maintaining less active (T) states of the kinase and the phosphatase domains. Phosphorylation of the peptide converts the kinase to a more active (R) state without affecting the phosphatase, but deletion of the peptide results in activation of the phosphatase to R state.
The mechanism of activation of heart fructose 6-phosphate,2-kinase: Fructose–2,6-bisphosphatase
Journal of Biological Chemistry
Partially purified fructose-6-P,2-kinase:fructose-2,6-bisphosphatase from beef heart was phosphorylated by cAMP protein kinase. The phosphorylated fructose-6-P,2-kinase shows lower Km for Fru-6-P (43 versus 105 microM) and for ATP (0.55 versus 1.3 mM) but no change in the Vmax, compared to those for unphosphorylated enzyme. There was no detectable change in Km or Vmax of fructose-2,6-bisphosphatase activity by the phosphorylation. These changes in heart fructose-6-P,2-kinase were in direct contrast to previous results for the liver isozyme in which phosphorylation led to inhibition of the kinase activity and activation of the phosphatase activity.
A rat gene encoding heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase
FEBS Letters, 1991
There are at least 3 isozymes of 6-phosphofructo-2-kinase/fructose-2,6bispl~ospha~se, a bifunctional enzyme which catalyzes the synthesis and degradation of fructose 2,6-bisphosphate. A 22-kb rat gene that encodes the heart isozyme has becr~ identified and compared with the 55kb rat gene encoding the liver and muscle isozymes which had been described earlier. Although these 2 genes include 12 successive similar exons, they contain dissimilar exons at both ends, consistent with the occurrence of different regulatory domains at the N-and C-termini in the 3 isozymes.
Journal of Biological Chemistry
Fructose-6-phosphate,2-kinase:fructose-2,6-bis-phosphatase from rat skeletal muscle has been purified to homogeneity, and its structure and kinetic properties have been determined. The Mr of the native enzyme was 100,000 and the subunit Mr was 54,000. The apparent Km values of fructose-6-P,2-kinase for Fru-6-P and ATP were 56 and 48 microM, respectively. The apparent Km value for Fru-2,6-P2 of fructose-2,6-bis-phosphatase was 0.4 microM, and the Ki for Fru-6-P was 12.5 microM. The enzyme was bifunctional, and the phosphatase activity was 2.5 times higher than the kinase activity. The enzyme was not phosphorylated by cAMP-dependent protein kinase. The amino acid composition of the skeletal muscle enzyme was similar to that of the rat liver enzyme, and the carboxyl terminus sequence (His-Tyr) was the same as that of the liver enzyme. The tryptic peptides generated from the liver and skeletal muscle enzymes were identical except for two peptides. A peptide corresponding to nucleotides ...