Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mechanism by which cAMP-dependent protein kinase-catalyzed phosphorylation modulates the activities of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was examined after site-specific mutation of the cAMP-dependent phosphorylation site (Ser32) to aspartic acid or alanine. The mutant and wild-type enzymes were overexpressed in Escherichia coli in a rich medium to levels as high as 30 mg/liter and were then purified to homogeneity. The kinetic properties of the Ser32-Ala mutant were identical with the dephosphorylated wild-type bifunctional enzyme. Mutation of Ser32 to aspartic acid mimicked several effects of cAMP-dependent phosphorylation: there was an increase in the Km for fructose 6-phosphate for 6-phosphofructo-2-kinase and an increase in the maximal velocity of fructose-2,6-bisphosphatase. Fructose-2,6-bisphosphatase activity of the Ser32-Asp mutant was 75% that of the phosphorylated wild-type enzyme, the mutant's kinase reaction had an identical dependence on fructose 6-phosphate, while its maximum velocity was only 60% that of the phosphorylated wild-type enzyme over a wide pH range. Furthermore, catalytic subunit-catalyzed in vitro phosphorylation of the Ser32-Ala mutant on Ser33 increased the Km for fructose 6-phosphate by 4-fold for the 6-phosphofructo-2-kinase. The results support the hypothesis that Ser32 is an important residue in the regulation of the activities of the bifunctional enzyme and that phosphorylation of Ser32 can be functionally substituted by aspartic acid. The results suggest a role for negative charge in the effect of phosphorylation.
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PMID:Rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Properties of phospho- and dephospho- forms and of two mutants in which Ser32 has been changed by site-directed mutagenesis. 133 50

We have isolated and sequenced two overlapping cDNA fragments which could encode the complete amino acid sequence of rat testis fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase. Northern blot analysis revealed that the major 2-kilobase mRNA isolated from rat testis hybridized with a cDNA fragment. A full length cDNA, which encoded a protein of 468 amino acids, was constructed and expressed in Escherichia coli. The expressed protein, purified to homogeneity, showed a Mr of 55,000 by gel electrophoresis under denaturing conditions, compared to the deduced Mr of 54,023. Fru-6-P,2-kinase:Fru-2,6-bisphosphatase with the same Mr 55,000 was also present in rat testis extract. The active enzyme was a dimer as judged by molecular sieve filtration. The expressed enzyme was bifunctional with specific activities of 90 and 22 milliunits/mg of the kinase and the phosphatase activities, respectively. Various kinetic constants of the expressed fructose 6-P,2-kinase were KmFru 6-P = 85 microM and KmATP = 270 microM, and those of fructose 2,6-bisphosphatase were KmFru 2,6-P2 = 21 microM and KiFru 6-P = 3.4 microM. The enzyme was phosphorylated by Fru-2,6[2-32P]P2 and also by protein kinase C, but not by cAMP-dependent protein kinase, which is in contrast to the liver and heart isozymes.
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PMID:Molecular cloning of the DNA and expression and characterization of rat testes fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase. 165 18

The effect of insulin on hepatic glucose production has been studied in anesthetized rats in the postabsorptive state. Insulin decreases significantly hepatic glucose production within 5-10 min. It also increases the level of fructose 2,6-bisphosphate, via an increase in the Vmax of 6-phosphofructo-2-kinase and concomitantly decreased the activity of fructose-2,6-bisphosphatase, resulting in a 5-fold increase in the ratio of kinase/phosphatase. Insulin also increased the apparent Kd of pyruvate kinase for phosphoenolpyruvate. The changes in the activity of 6-phosphofructo-2-kinase and pyruvate kinase were measured after separation from possible modulators, and suggest a decrease in their phosphorylation state which cannot be attributed to a decrease in the level of cAMP or in the activity of cAMP-dependent protein kinase since these two parameters were not modified by insulin. In addition, neither the activity of phosphorylase a nor that of glycogen synthase were modified. The data strongly suggest that the increase in the glycolytic rate plays a role in the effect of insulin on hepatic glucose production and that insulin mediates its effect on the activity of these enzymes via one or more phosphatases.
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PMID:Insulin activates 6-phosphofructo-2-kinase and pyruvate kinase in the liver. Indirect evidence for an action via a phosphatase. 215 92

Hormonal regulation of hepatic gluconeogenic pathway flux is brought about by phosphorylation/dephosphorylation and control of gene expression of several key regulatory enzymes. Regulation by cAMP-dependent phosphorylation occurs at the level of pyruvate kinase and 6-phosphofructo-2-kinase (6PF-1-K)/fructose-2,6-bisphosphatase (Fru-2,6-P2ase). The latter is a unique bifunctional enzyme that catalyzes both the synthesis and degradation of fructose-2,6-bisphosphate (Fru-2,6-P2), which is an activator of 6PF-1-K and an inhibitor of Fru-1,6-P2ase. The bifunctional enzyme is a homodimer whose activities are regulated by cAMP-dependent protein kinase-catalyzed phosphorylation at a single NH2-terminal seryl residue/subunit, which results in activation of the Fru-2,6-P2ase and inhibition of the PF-1-K reactions. Hormone-mediated changes in the phosphorylation state of the bifunctional enzyme are responsible for acute regulation of Fru-2,6-P2 levels. 6PF-2-K/Fru-2,6-P2ase thus provides a switching mechanism between glycolysis and gluconeogenesis in mammalian liver. Pyruvate kinase is regulated by both phosphorylation and allosteric effectors. Fru-1,6-P2, an allosteric activator, also inhibits cAMP-dependent enzyme phosphorylation, and its steady-state concentration is indirectly determined by the level of Fru-2,6-P2. Therefore, acute regulation of both pyruvate kinase and the bifunctional enzyme provide coordinated control at both the pyruvate/phosphoenolpyruvate and Fru-6-P/Fru-1,6-P2 substrate cycles. The Fru-2,6-P2 system is also subject to complex multihormonal long-term control through regulation of 6 PF-2-K/Fru-2,6-P2ase gene expression. Glucocorticoids are the major factor in turning on this gene in liver, but insulin is also a positive effector. cAMP prevents the effects of glucocorticoids and insulin. Although Fru-2,6-P2 plays a key role in the regulation of carbon flux in the gluconeogenic pathway, the regulation of this flux depends on several factors and regulation of other key enzymes whose importance varies depending on the dietary and hormonal status of the animal. Molecular cloning of the cDNA encoding PF-2-K/Fru-2,6-P2ase has elucidated its structure and permitted analysis of its evolutionary origin as well as its tissue distribution and control of its gene expression. The rat liver and skeletal muscle isoforms arose by alternative splicing of a single gene. The muscle form differs from the liver form only at the NH2-terminal and does not have a cAMP-dependent protein kinase phosphorylation site. The hepatic enzyme subunit consists of 470 amino acids.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Fructose-2,6-bisphosphate in control of hepatic gluconeogenesis. From metabolites to molecular genetics. 216 55

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 cAMP-dependent 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.
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PMID:Bovine heart fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase: complete amino acid sequence and localization of phosphorylation sites. 216 12

The synthesis and degradation of fructose 2,6-bisphosphate, a ubiquitous stimulator of glycolysis, are catalyzed by 6-phosphofructo-2-kinase (EC 2.7.1.105) and fructose-2,6-bisphosphatase (EC 3.1.3.46), respectively. In liver, these two activities belong to separate domains of the same 470-residue polypeptide. Various mRNAs have been described for this bifunctional enzyme, which is controlled by hormonal and metabolic signals. To understand the origin and regulation of these mRNAs, we have characterized rat genomic clones encoding the liver isozyme, which is regulated by cAMP-dependent protein kinase, and the muscle isozyme, which is not. We describe here a 55-kilobase gene that encodes these isozymes by alternative splicing from two promoters. Each of the putative promoters was sequenced over about 3 kilobases and found to include nucleotide motifs for binding regulatory factors. The two isozymes share the same 13 exons and differ only by the first exon that, in the liver but not in the muscle isozyme, contains the serine phosphorylated by cAMP-dependent protein kinase. The gene was assigned to the X chromosome. An analysis of the exon limits of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in relation to its functional domains and to its similarity with other proteins plus its G + C content at the third codon position suggests that this gene originates from several fusion events.
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PMID:5' flanking sequence and structure of a gene encoding rat 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. 254 41

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 Mr 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,2-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 Km of the kinase for fructose-6-P and ATP was 70 microM and 260 microM, respectively for both the 58,000 and the 54,000 enzymes. Km for fructose-2,6-P2 and Ki for fructose-6-P of the phosphatase was approximately 40 and 11 microM, respectively. The enzyme was phosphorylated by fructose-2,6-P2 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.
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PMID:Purification and characterization of myocardial fructose-6-phosphate,2-kinase and fructose-2,6-bisphosphatase. 283 72

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 Vmax, whereas the phosphorylation by protein kinase C increased the Vmax value from 55 to 85 milliunits/mg without altering the Km value. The phosphorylated peptides were isolated, and their amino acid sequences were determined. The phosphorylation sites for both cAMP-dependent protein kinase and protein kinase C were located in a single peptide whose sequence was Arg-Arg-Asn-Ser-(P)-Phe-Thr-Pro-Leu-Ser-Ser-Ser-Asn-Thr(P)-Ile-Arg-Arg-Pro. The seryl residue nearest the N terminus was the residue specifically phosphorylated by cAMP-dependent protein kinase, whereas the threonine residue nearest the C terminus was phosphorylated by protein kinase C.
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PMID:Phosphorylation of myocardial fructose-6-phosphate,2-kinase: fructose-2,6-bisphosphatase by cAMP-dependent protein kinase and protein kinase C. Activation by phosphorylation and amino acid sequences of the phosphorylation sites. 284 51

6-Phosphofructo-2-kinase and fructose-2,6-bisphosphatase activities were copurified to homogeneity from bovine liver. The purification scheme consisted of polyethylene glycol precipitation, anion-exchange and Blue-Sepharose chromatography, substrate elution from phosphocellulose, and gel filtration. The bifunctional enzyme had an apparent molecular weight of 102,000 and consisted of two subunits (Mr 49,000). The kinase had a Km for ATP of 12 microM and a S0.5 for fructose 6-phosphate of 150 microM while the bisphosphatase had a Km for fructose 2,6-bisphosphate of 7 microM. Both activities were subject to modulation by various effectors. Inorganic phosphate stimulated both activities, while alpha-glycerolphosphate inhibited the kinase and stimulated the bisphosphatase. The pH optimum for the 6-phosphofructo-2-kinase activity was 8.5, while the fructose-2,6-bisphosphatase reaction was maximal at pH 6.5. Incubation of the purified enzyme with [gamma-32P]ATP and the catalytic subunit of the cAMP-dependent protein kinase resulted in 32P incorporation to the extent of 0.7 mol/mol enzyme subunit with concomitant inhibition of the kinase activity and activation of the bisphosphatase activity. The mediation of the bisphosphatase reaction by a phosphoenzyme intermediate was suggested by the isolation of a stable labeled phosphoenzyme when the enzyme was incubated with fructose 2,6-[2-32P]bisphosphate. The pH dependence of hydrolysis of the phospho group suggested that it was linked to the N3 of a histidyl residue. The 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from bovine liver has properties essentially identical to those of the rat liver enzyme, suggesting that hepatic fructose 2,6-bisphosphate metabolism is under the same control in both species.
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PMID:Isolation and characterization of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from bovine liver. 298 53

The bisphosphatase reaction sequence of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase involves a phosphoenzyme intermediate. Catalysis is activated in vitro by cAMP-dependent protein kinase-catalyzed phosphorylation. We investigated the mechanism of this activation by studying the effect of protein kinase-catalyzed phosphorylation on the formation and breakdown of the phosphoenzyme intermediate. The significant findings were as follows. 1) Phosphorylation decreased the rate of phosphoenzyme formation. 2) More importantly, phosphorylation increased the much slower rate of phosphoenzyme breakdown both in the absence and presence of the regulatory ligands, inorganic phosphate and alpha-glycerol phosphate. The increase in the rate of phosphoenzyme breakdown correlated with the degree of activation of the bisphosphatase; both were increased about 2-fold. 3) The potent inhibition of phosphoenzyme breakdown by fructose 6-phosphate indicates that, in the catalytic sequence, the release of nascent fructose 6-phosphate from the active site precedes phosphoenzyme breakdown and Pi release. 4) Phosphorylation reduced the fructose 6-phosphate inhibition of phosphoenzyme breakdown both in the absence and presence of phosphate and alpha-glycerol phosphate. 5) Phosphorylation decreased the potent substrate inhibition which occurs at physiological substrate concentrations. It appears that protein kinase-catalyzed phosphorylation activates fructose-2,6-bisphosphatase by promoting the dissociation of fructose 6-phosphate and fructose 2,6-bisphosphate from the same phosphoenzyme intermediate, hastening its exposure to water and thereby relieving both product and substrate inhibitions.
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PMID:Mechanism of activation of fructose-2,6-bisphosphatase by cAMP-dependent protein kinase. 301 63


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