Skip to main content
NCBI home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1995 Jun;4(6):1023–1037. doi: 10.1002/pro.5560040601

Covalent control of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: insights into autoregulation of a bifunctional enzyme.

I J Kurland 1, S J Pilkis 1
PMCID: PMC2143155  PMID: 7549867

Abstract

The hepatic bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF-2-K/Fru-2,6-P2ase), E.C. 2.7-1-105/E.C. 3-1-3-46, is one member of a family of unique bifunctional proteins that catalyze the synthesis and degradation of the regulatory metabolite fructose-2,6-bisphosphate (Fru-2,6-P2). Fru-2,6-P2 is a potent activator of the glycolytic enzyme 6-phosphofructo-1-kinase and an inhibitor of the gluconeogenic enzyme fructose-1,6-bisphosphatase, and provides a switching mechanism between these two opposing pathways of hepatic carbohydrate metabolism. The activities of the hepatic 6PF-2-K/Fru-2,6-P2ase isoform are reciprocally regulated by a cyclic AMP-dependent protein kinase (cAPK)-catalyzed phosphorylation at a single NH2-terminal residue, Ser-32. Phosphorylation at Ser-32 inhibits the kinase and activates the bisphosphatase, in part through an electrostatic mechanism. Substitution of Asp for Ser-32 mimics the effects of cAPK-catalyzed phosphorylation. In the dephosphorylated homodimer, the NH2- and COOH-terminal tail regions also have an interaction with their respective active sites on the same subunit to produce an autoregulatory inhibition of the bisphosphatase and activation of the kinase. In support of this hypothesis, deletion of either the NH2- or COOH-terminal tail region, or both regions, leads to a disruption of these interactions with a maximal activation of the bisphosphatase. Inhibition of the kinase is observed with the NH2-truncated forms, in which there is also a diminution of cAPK phosphorylation to decrease the Km for Fru-6-P. Phosphorylation of the bifunctional enzyme by cAPK disrupts these autoregulatory interactions, resulting in inhibition of the kinase and activation of the bisphosphatase. Therefore, effects of cyclic AMP-dependent phosphorylation are mediated by a combination of electrostatic and autoregulatory control mechanisms.

Full Text

The Full Text of this article is available as a PDF (4.4 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bazan J. F., Fletterick R. J., Pilkis S. J. Evolution of a bifunctional enzyme: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9642–9646. doi: 10.1073/pnas.86.24.9642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Becker J. U., Wingender-Drissen R., Schiltz E. Purification and properties of phosphorylase from baker's yeast. Arch Biochem Biophys. 1983 Sep;225(2):667–678. doi: 10.1016/0003-9861(83)90078-4. [DOI] [PubMed] [Google Scholar]
  3. Benkovic S. J. Catalytic antibodies. Annu Rev Biochem. 1992;61:29–54. doi: 10.1146/annurev.bi.61.070192.000333. [DOI] [PubMed] [Google Scholar]
  4. Berger S. A., Evans P. R. Active-site mutants altering the cooperativity of E. coli phosphofructokinase. Nature. 1990 Feb 8;343(6258):575–576. doi: 10.1038/343575a0. [DOI] [PubMed] [Google Scholar]
  5. Brändeén C. I. Relation between structure and function of alpha/beta-proteins. Q Rev Biophys. 1980 Aug;13(3):317–338. doi: 10.1017/s0033583500001712. [DOI] [PubMed] [Google Scholar]
  6. Crepin K. M., De Cloedt M., Vertommen D., Foret D., Michel A., Rider M. H., Rousseau G. G., Hue L. Molecular forms of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase expressed in rat skeletal muscle. J Biol Chem. 1992 Oct 25;267(30):21698–21704. [PubMed] [Google Scholar]
  7. Darville M. I., Crepin K. M., Hue L., Rousseau G. G. 5' flanking sequence and structure of a gene encoding rat 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Proc Natl Acad Sci U S A. 1989 Sep;86(17):6543–6547. doi: 10.1073/pnas.86.17.6543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Darville M. I., Crepin K. M., Vandekerckhove J., Van Damme J., Octave J. N., Rider M. H., Marchand M. J., Hue L., Rousseau G. G. Complete nucleotide sequence coding for rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase derived from a cDNA clone. FEBS Lett. 1987 Nov 30;224(2):317–321. doi: 10.1016/0014-5793(87)80476-3. [DOI] [PubMed] [Google Scholar]
  9. Dean A. M., Koshland D. E., Jr Electrostatic and steric contributions to regulation at the active site of isocitrate dehydrogenase. Science. 1990 Aug 31;249(4972):1044–1046. doi: 10.1126/science.2204110. [DOI] [PubMed] [Google Scholar]
  10. Depre C., Rider M. H., Veitch K., Hue L. Role of fructose 2,6-bisphosphate in the control of heart glycolysis. J Biol Chem. 1993 Jun 25;268(18):13274–13279. [PubMed] [Google Scholar]
  11. El-Maghrabi M. R., Claus T. H., Pilkis J., Fox E., Pilkis S. J. Regulation of rat liver fructose 2,6-bisphosphatase. J Biol Chem. 1982 Jul 10;257(13):7603–7607. [PubMed] [Google Scholar]
  12. El-Maghrabi M. R., Claus T. H., Pilkis J., Pilkis S. J. Partial purification of a rat liver enzyme that catalyzes the formation of fructose 2,6-bisphosphate. Biochem Biophys Res Commun. 1981 Aug 14;101(3):1071–1077. doi: 10.1016/0006-291x(81)91858-1. [DOI] [PubMed] [Google Scholar]
  13. El-Maghrabi M. R., Fox E., Pilkis J., Pilkis S. J. Cyclic AMP-dependent phosphorylation of rat liver 6-phosphofructo 2-kinase, fructose 2,6-bisphosphatase. Biochem Biophys Res Commun. 1982 Jun 15;106(3):794–802. doi: 10.1016/0006-291x(82)91780-6. [DOI] [PubMed] [Google Scholar]
  14. El-Maghrabi M. R., Pate T. M., Murray K. J., Pilkis S. J. Differential effects of proteolysis and protein modification on the activities of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. J Biol Chem. 1984 Nov 10;259(21):13096–13103. [PubMed] [Google Scholar]
  15. El-Maghrabi M. R., Pate T. M., Pilkis S. J. Characterization of the exchange reactions of rat liver 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase. Biochem Biophys Res Commun. 1984 Sep 17;123(2):749–756. doi: 10.1016/0006-291x(84)90293-6. [DOI] [PubMed] [Google Scholar]
  16. Fothergill-Gilmore L. A., Watson H. C. The phosphoglycerate mutases. Adv Enzymol Relat Areas Mol Biol. 1989;62:227–313. doi: 10.1002/9780470123089.ch6. [DOI] [PubMed] [Google Scholar]
  17. Hu S. H., Parker M. W., Lei J. Y., Wilce M. C., Benian G. M., Kemp B. E. Insights into autoregulation from the crystal structure of twitchin kinase. Nature. 1994 Jun 16;369(6481):581–584. doi: 10.1038/369581a0. [DOI] [PubMed] [Google Scholar]
  18. Hurley J. H., Dean A. M., Sohl J. L., Koshland D. E., Jr, Stroud R. M. Regulation of an enzyme by phosphorylation at the active site. Science. 1990 Aug 31;249(4972):1012–1016. doi: 10.1126/science.2204109. [DOI] [PubMed] [Google Scholar]
  19. Johnson L. N. Control by protein phosphorylation. Nat Struct Biol. 1994 Oct;1(10):657–659. doi: 10.1038/nsb1094-657. [DOI] [PubMed] [Google Scholar]
  20. Johnson L. N. Glycogen phosphorylase: control by phosphorylation and allosteric effectors. FASEB J. 1992 Mar;6(6):2274–2282. doi: 10.1096/fasebj.6.6.1544539. [DOI] [PubMed] [Google Scholar]
  21. Kemp B. E., Parker M. W., Hu S., Tiganis T., House C. Substrate and pseudosubstrate interactions with protein kinases: determinants of specificity. Trends Biochem Sci. 1994 Nov;19(11):440–444. doi: 10.1016/0968-0004(94)90126-0. [DOI] [PubMed] [Google Scholar]
  22. Kemp B. E., Pearson R. B. Intrasteric regulation of protein kinases and phosphatases. Biochim Biophys Acta. 1991 Aug 13;1094(1):67–76. doi: 10.1016/0167-4889(91)90027-u. [DOI] [PubMed] [Google Scholar]
  23. Kitajima S., Sakakibara R., Uyeda K. Kinetic studies of fructose 6-phosphate,2-kinase and fructose 2,6-bisphosphatase. J Biol Chem. 1984 Jun 10;259(11):6896–6903. [PubMed] [Google Scholar]
  24. Kitamura K., Kangawa K., Matsuo H., Uyeda K. 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. J Biol Chem. 1988 Nov 15;263(32):16796–16801. [PubMed] [Google Scholar]
  25. Kitamura K., Uyeda K., Kangawa K., Matsuo H. Purification and characterization of rat skeletal muscle fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase. J Biol Chem. 1989 Jun 15;264(17):9799–9806. [PubMed] [Google Scholar]
  26. Kitamura K., Uyeda K. The mechanism of activation of heart fructose 6-phosphate,2-kinase:fructose-2,6-bisphosphatase. J Biol Chem. 1987 Jan 15;262(2):679–681. [PubMed] [Google Scholar]
  27. Knighton D. R., Pearson R. B., Sowadski J. M., Means A. R., Ten Eyck L. F., Taylor S. S., Kemp B. E. Structural basis of the intrasteric regulation of myosin light chain kinases. Science. 1992 Oct 2;258(5079):130–135. doi: 10.1126/science.1439761. [DOI] [PubMed] [Google Scholar]
  28. Knowles J. R. Enzyme-catalyzed phosphoryl transfer reactions. Annu Rev Biochem. 1980;49:877–919. doi: 10.1146/annurev.bi.49.070180.004305. [DOI] [PubMed] [Google Scholar]
  29. Kountz P. D., Freeman S., Cook A. G., el-Maghrabi M. R., Knowles J. R., Pilkis S. J. The stereochemical course of phospho group transfer catalyzed by rat liver 6-phosphofructo-2-kinase. J Biol Chem. 1988 Nov 5;263(31):16069–16072. [PubMed] [Google Scholar]
  30. Kountz P. D., McCain R. W., el-Maghrabi M. R., Pilkis S. J. Hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: phosphate dependence and effects of other oxyanions. Arch Biochem Biophys. 1986 Nov 15;251(1):104–113. doi: 10.1016/0003-9861(86)90056-1. [DOI] [PubMed] [Google Scholar]
  31. Kretschmer M., Fraenkel D. G. Yeast 6-phosphofructo-2-kinase: sequence and mutant. Biochemistry. 1991 Nov 5;30(44):10663–10672. doi: 10.1021/bi00108a009. [DOI] [PubMed] [Google Scholar]
  32. Kurland I. J., Li L., Lange A. J., Correia J. J., el-Maghrabi M. R., Pilkis S. J. Regulation of rat 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Role of the NH2-terminal region. J Biol Chem. 1993 Jul 5;268(19):14056–14064. [PubMed] [Google Scholar]
  33. Kurland I. J., el-Maghrabi M. R., Correia J. J., Pilkis S. J. 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. J Biol Chem. 1992 Mar 5;267(7):4416–4423. [PubMed] [Google Scholar]
  34. Lange A. J., Pilkis S. J. Sequence of human liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Nucleic Acids Res. 1990 Jun 25;18(12):3652–3652. doi: 10.1093/nar/18.12.3652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Lange A. J., el-Maghrabi M. R., Pilkis S. J. Isolation of bovine liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase cDNA: bovine liver and heart forms of the enzyme are separate gene products. Arch Biochem Biophys. 1991 Oct;290(1):258–263. doi: 10.1016/0003-9861(91)90617-r. [DOI] [PubMed] [Google Scholar]
  36. Lei J., Tang X., Chambers T. C., Pohl J., Benian G. M. Protein kinase domain of twitchin has protein kinase activity and an autoinhibitory region. J Biol Chem. 1994 Aug 19;269(33):21078–21085. [PubMed] [Google Scholar]
  37. Li L., Lange A. J., Pilkis S. J. Isolation of a cDNA for chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Biochem Biophys Res Commun. 1993 Jan 29;190(2):397–405. doi: 10.1006/bbrc.1993.1061. [DOI] [PubMed] [Google Scholar]
  38. Li L., Lin K., Correia J. J., Pilkis S. J. Lysine 356 is a critical residue for binding the C-6 phospho group of fructose 2,6-bisphosphate to the fructose-2,6-bisphosphatase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. J Biol Chem. 1992 Aug 15;267(23):16669–16675. [PubMed] [Google Scholar]
  39. Li L., Lin K., Pilkis J., Correia J. J., Pilkis S. J. Hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. The role of surface loop basic residues in substrate binding to the fructose-2,6-bisphosphatase domain. J Biol Chem. 1992 Oct 25;267(30):21588–21594. [PubMed] [Google Scholar]
  40. Lin K., Kurland I. J., Li L., Lee Y. H., Okar D., Marecek J. F., Pilkis S. J. Evidence for NH2- and COOH-terminal interactions in rat 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. J Biol Chem. 1994 Jun 17;269(24):16953–16960. [PubMed] [Google Scholar]
  41. Lin K., Li L., Correia J. J., Pilkis S. J. Arg-257 and Arg-307 of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase bind the C-2 phospho group of fructose-2,6-bisphosphate in the fructose-2,6-bisphosphatase domain. J Biol Chem. 1992 Sep 25;267(27):19163–19171. [PubMed] [Google Scholar]
  42. Lin K., Li L., Correia J. J., Pilkis S. J. Glu327 is part of a catalytic triad in rat liver fructose-2,6-bisphosphatase. J Biol Chem. 1992 Apr 5;267(10):6556–6562. [PubMed] [Google Scholar]
  43. Paravicini G., Kretschmer M. The yeast FBP26 gene codes for a fructose-2,6-bisphosphatase. Biochemistry. 1992 Aug 11;31(31):7126–7133. doi: 10.1021/bi00146a014. [DOI] [PubMed] [Google Scholar]
  44. Pilkis S. J., Claus T. H. Hepatic gluconeogenesis/glycolysis: regulation and structure/function relationships of substrate cycle enzymes. Annu Rev Nutr. 1991;11:465–515. doi: 10.1146/annurev.nu.11.070191.002341. [DOI] [PubMed] [Google Scholar]
  45. Pilkis S. J., Lively M. O., el-Maghrabi M. R. Active site sequence of hepatic fructose-2,6-bisphosphatase. Homology in primary structure with phosphoglycerate mutase. J Biol Chem. 1987 Sep 15;262(26):12672–12675. [PubMed] [Google Scholar]
  46. Pilkis S. J., Pilkis J., el-Maghrabi M. R., Claus T. H. The sugar phosphate specificity of rat hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. J Biol Chem. 1985 Jun 25;260(12):7551–7556. [PubMed] [Google Scholar]
  47. Rath V. L., Fletterick R. J. Parallel evolution in two homologues of phosphorylase. Nat Struct Biol. 1994 Oct;1(10):681–690. doi: 10.1038/nsb1094-681. [DOI] [PubMed] [Google Scholar]
  48. Rider M. H., Vandamme J., Lebeau E., Vertommen D., Vidal H., Rousseau G. G., Vandekerckhove J., Hue L. The two forms of bovine heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase result from alternative splicing. Biochem J. 1992 Jul 15;285(Pt 2):405–411. doi: 10.1042/bj2850405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Sakata J., Abe Y., Uyeda K. Molecular cloning of the DNA and expression and characterization of rat testes fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase. J Biol Chem. 1991 Aug 25;266(24):15764–15770. [PubMed] [Google Scholar]
  50. Sakata J., Uyeda K. Bovine heart fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase: complete amino acid sequence and localization of phosphorylation sites. Proc Natl Acad Sci U S A. 1990 Jul;87(13):4951–4955. doi: 10.1073/pnas.87.13.4951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Schirmer T., Evans P. R. Structural basis of the allosteric behaviour of phosphofructokinase. Nature. 1990 Jan 11;343(6254):140–145. doi: 10.1038/343140a0. [DOI] [PubMed] [Google Scholar]
  52. Shirakihara Y., Evans P. R. Crystal structure of the complex of phosphofructokinase from Escherichia coli with its reaction products. J Mol Biol. 1988 Dec 20;204(4):973–994. doi: 10.1016/0022-2836(88)90056-3. [DOI] [PubMed] [Google Scholar]
  53. Soderling T. R. Protein kinases. Regulation by autoinhibitory domains. J Biol Chem. 1990 Feb 5;265(4):1823–1826. [PubMed] [Google Scholar]
  54. Stewart H. B., el-Maghrabi M. R., Pilkis S. J. Mechanism of activation of fructose-2,6-bisphosphatase by cAMP-dependent protein kinase. J Biol Chem. 1986 Jul 5;261(19):8793–8798. [PubMed] [Google Scholar]
  55. Tauler A., Lin K., Pilkis S. J. Hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Use of site-directed mutagenesis to evaluate the roles of His-258 and His-392 in catalysis. J Biol Chem. 1990 Sep 15;265(26):15617–15622. [PubMed] [Google Scholar]
  56. Tominaga N., Minami Y., Sakakibara R., Uyeda K. Significance of the amino terminus of rat testis fructose-6-phosphate, 2-kinase:fructose-2,6-bisphosphatase. J Biol Chem. 1993 Jul 25;268(21):15951–15957. [PubMed] [Google Scholar]
  57. Tsuchiya Y., Uyeda K. Bovine heart fructose 6-P,2-kinase:fructose 2,6-bisphosphatase mRNA and gene structure. Arch Biochem Biophys. 1994 May 1;310(2):467–474. doi: 10.1006/abbi.1994.1194. [DOI] [PubMed] [Google Scholar]
  58. Valdez B. C., French B. A., Younathan E. S., Chang S. H. Site-directed mutagenesis in Bacillus stearothermophilus fructose-6-phosphate 1-kinase. Mutation at the substrate-binding site affects allosteric behavior. J Biol Chem. 1989 Jan 5;264(1):131–135. [PubMed] [Google Scholar]
  59. Van Schaftingen E., Davies D. R., Hers H. G. Inactivation of phosphofructokinase 2 by cyclic AMP - dependent protein kinase. Biochem Biophys Res Commun. 1981 Nov 16;103(1):362–368. doi: 10.1016/0006-291x(81)91701-0. [DOI] [PubMed] [Google Scholar]
  60. Van Schaftingen E., Hers H. G. Purification and properties of phosphofructokinase 2/fructose 2,6-bisphosphatase from chicken liver and from pigeon muscle. Eur J Biochem. 1986 Sep 1;159(2):359–365. doi: 10.1111/j.1432-1033.1986.tb09876.x. [DOI] [PubMed] [Google Scholar]
  61. Ventura F., Rosa J. L., Ambrosio S., Pilkis S. J., Bartrons R. Bovine brain 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Evidence for a neural-specific isozyme. J Biol Chem. 1992 Sep 5;267(25):17939–17943. [PubMed] [Google Scholar]
  62. Winn S. I., Watson H. C., Harkins R. N., Fothergill L. A. Structure and activity of phosphoglycerate mutase. Philos Trans R Soc Lond B Biol Sci. 1981 Jun 26;293(1063):121–130. doi: 10.1098/rstb.1981.0066. [DOI] [PubMed] [Google Scholar]
  63. Zheng J., Knighton D. R., ten Eyck L. F., Karlsson R., Xuong N., Taylor S. S., Sowadski J. M. Crystal structure of the catalytic subunit of cAMP-dependent protein kinase complexed with MgATP and peptide inhibitor. Biochemistry. 1993 Mar 9;32(9):2154–2161. doi: 10.1021/bi00060a005. [DOI] [PubMed] [Google Scholar]
  64. el-Maghrabi M. R., Gidh-Jain M., Austin L. R., Pilkis S. J. Isolation of a human liver fructose-1,6-bisphosphatase cDNA and expression of the protein in Escherichia coli. Role of ASP-118 and ASP-121 in catalysis. J Biol Chem. 1993 May 5;268(13):9466–9472. [PubMed] [Google Scholar]
  65. van Schaftingen E., Davies D. R., Hers H. G. Fructose-2,6-bisphosphatase from rat liver. Eur J Biochem. 1982 May;124(1):143–149. doi: 10.1111/j.1432-1033.1982.tb05917.x. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES