The mechanism of activation of heart fructose 6-phosphate,2-kinase: Fructose–2,6-bisphosphatase (original) (raw)
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Journal of Biological Chemistry
Phosphorylation of pure fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase from bovine heart by cAMP-dependent protein kinase and protein kinase C was investigated. The major enzyme form (subunit Mr of 58,000) was rapidly phosphorylated by both cAMP-dependent protein kinase and protein kinase C, incorporating 0.8 and 1.0 mol/mol of subunit, respectively. The rate of phosphorylation of the heart enzyme by cAMP-dependent protein kinase was 10 times faster than that of the rat liver enzyme. The minor enzyme (subunit Mr of 54,000), however, was phosphorylated only by protein kinase C and was phosphorylated much more slowly with a phosphate incorporation of less than 0.1 mol/mol of subunit. Phosphorylation by either cAMP-dependent protein kinase or protein kinase C activated the enzyme, but each phosphorylation affected different kinetic parameters. Phosphorylation by cAMP-dependent protein kinase lowered the Km value for fructose 6-phosphate from 87 to 42 microM without affecting...
The Journal of Biological Chemistry, 1988
Phosphorylation of pure fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase from bovine heart by CAMP-dependent protein kinase and protein kinase C was investigated. The major enzyme form (subunit M. of 58,000) was rapidly phosphorylated by both CAMPdependent protein kinase and protein kinase C, incorporating 0.8 and 1.0 mol/mol of subunit, respectively. The rate of phosphorylation of the heart enzyme by CAMP-dependent protein kinase was 10 times faster than that of the rat liver enzyme. The minor enzyme (subunit M, of 54,000), however, was phosphorylated only by protein kinase C and was phosphorylated much more slowly with a phosphate incorporation of less than 0.1 mol/mol of subunit. Phosphorylation by either CAMP-dependent protein kinase or protein kinase C activated the enzyme, but each phosphorylation affected different kinetic parameters. Phosphorylation by CAMP-dependent protein kinase lowered the K, value for fructose 6-phosphate from 87 to 42 p~ without affecting the V, , , whereas the phosphorylation by protein kinase C increased the V,, value from 55 to 85 milliunits/mg without altering the K, value.
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.
Biochemical and Biophysical Research Communications, 1991
Occurrence of two isozymic forms of fructose 6-P,2-kinase: fructose 2,6-bisphosphatase in bovine heart was investigated by transcribing mRNAs and amplifying the cDNAs with polymerase chain reactions. Analysis of the PCR products revealed 1.7 Kb and 1.5 Kb DNAs, and the determination of their nucleotide sequences showed that these DNAs are identical except for the lack of 180 base pairs near the 3' of the bovine heart enzyme DNA previously reported (6). This missing nucleotide sequence encodes Asn4S1-Gln~° and contains the phosphorylation sites for cAMP dependent protein kinase and protein kinase C.
Journal of Biological Chemistry, 1988
Fructose-6-P,2-kinase:fructose-2,6-bisphosphatase has been purified to homogeneity from beef heart. The enzyme was bifunctional and the specific activities of the kinase and the phosphatase of the pure enzyme were 60 and 30 milliunits/mg, respectively. The molecular weight of the enzyme was 118,000, consisting of two subunits of 58,000. In some preparations of the enzyme a minor protein with a subunit M, of 54,000 was present. This minor protein (54,000) was also bifunctional and showed the same immunoreactivity as the major protein. The specific activity of fructose-6-P,a-kinase of the minor component was three times higher than that of the major enzyme (58,000), but fructose-2,6-bisphosphatase activity was the same. These two forms have been separated by phosphocellulose chromatography. The tryptic peptide maps of these enzymes were very similar. The 58,000 enzyme was phosphorylated by CAMP-dependent protein kinase but the 54,000 enzyme was not. These results indicated that the minor 54,000 protein might be a proteolytically digested form of the 58,000 enzyme. The K, of the kinase for fructose-6-P and ATP was 70 PM and 260 PM, respectively for both the 58,000 and the 54,000 enzymes. K,,, for fructose-2,6-P2 and Ki for fructose-6-P of the phosphatase was approximately 40 and 11 PM, respectively. The enzyme was phosphorylated by fructose-2,6-P~ but the stoichiometry of the phosphate incorporation was 0.05 mol/mol subunit, while 0.4 mol/mol was incorporated in rat liver enzyme under the same conditions.
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.
Journal of Biological Chemistry
Fructose-6-P binding sites of rat liver and bovine heart Fru-6-P,2-kinase:Fru-2,6-bisphosphatase were investigated with an affinity labeling reagent, N-bromoacetylethanolamine phosphate. The rat liver enzyme was inactivated 97% by the reagent in 60 min, and the rate of inactivation followed pseudo-first order kinetics. The bovine heart enzyme was inactivated 90% within 60 min, but the inactivation rate followed pseudo-first order up to 80% inactivation and then became nonlinear. The presence of fructose-6-P retarded the extent of the inactivation to approximately 40% in 60 min. In order to determine the amino acid sequence of the fructose-6-P binding site, both enzymes were reacted with N-bromo[14C]acetylethanolamine-P and digested with trypsin; radiolabeled tryptic peptides were isolated and sequenced. A single 14C-labeled peptide was isolated from the rat liver enzyme, and the amino acid sequence of the peptide was determined as Lys-Gln-Cys-Ala-Leu-Ala-Leu-Lys. A major and two min...
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 ...
Role of fructose 2,6-bisphosphate in the control of heart glycolysis
Journal of Biological Chemistry
The aim of this work was to study whether changes in fructose 2,6-bisphosphate concentration are correlated with variations of the glycolytic flux in the isolated working rat heart. Glycolysis was stimulated to different extents by increasing the concentration of glucose, increasing the workload, or by the addition of insulin. The glycolytic flux was measured by the rate of detritiation of [2-'H]-and [3-'H]glucose. Under all the conditions tested, an increase in fructose 2,6-bisphosphate content was observed. The glucose-or insulin-induced increase in fructose 2,6-bisphosphate content was related to an increase in the concentration of fructose 6-phosphate, the substrate of 6-phosphofructo-2-kinase. An increase in the workload correlated with a 50% decrease in the K,,, of 6-phosphofructo-2-kinase for fructose 6-phosphate. Similar changes in K,,, have been observed when purified heart 6-phosphofructo-2-kinase was phosphorylated in vitro by the cyclic AMP-dependent protein kinase or by the calcium/calmodulin-dependent protein kinase. Since the concentration of cyclic AMP was not affected by increasing the workload, it is possible that the change in K,,, of 6-phosphofructo-2-kinase, which was found in hearts submitted to a high load, resulted from phosphorylation by calcium/calmodulin protein kinase; other possibilities are not excluded. Anoxia decreased the external work developed by the heart, stimulated glycolysis and glycogenolysis, but did not increase fructose 2,6-bisphosphate.