Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1997 Feb 1;321(Pt 3):623–627. doi: 10.1042/bj3210623

Site-directed mutagenesis of Lys-174, Asp-179 and Asp-191 in the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

L Bertrand 1, J Deprez 1, D Vertommen 1, A Di Pietro 1, L Hue 1, M H Rider 1
PMCID: PMC1218115  PMID: 9032446

Abstract

In a structural model of the 2-kinase domain of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase based on the analogy with adenylate kinase, Lys-174, Asp-179 and Asp-191 residues are located in the putative active site. Asp-179 and Asp-191 are conserved in all known 6-phosphofructo-2-kinase sequences. In contrast, Lys-174 is conserved except in a yeast isoenzyme, fbp26, where it is replaced by glycine. Yeast fbp26 possesses fructose-2,6-bisphosphatase activity, but is devoid of 6-phosphofructo-2-kinase activity. Mutation of Asp-179 and Asp-191 of the rat liver isoenzyme to alanine increased the Km of 6-phosphofructo-2-kinase for fructose 6-phosphate 2000- and 1000-fold respectively, whereas mutation of Lys-174 to glycine decreased the Vmax of 6-phosphofructo-2-kinase more than 4000-fold. In contrast, none of the mutations affected the kinetic parameters of fructose-2,6-bisphosphatase. CD and fluorescence measurements indicated that the mutations had no effect on the structure and stability of the recombinant proteins. The results show that Asp-179 and Asp-191 participate in fructose 6-phosphate binding, whereas Lys-174 is important for catalysis. Therefore the natural mutation of Lys-174 to glycine in the fbp26 yeast isoenzyme could explain the lack of 6-phosphofructo-2-kinase activity. These results support a novel 6-phosphofructo-2-kinase structure model based on adenylate kinase.

Full Text

The Full Text of this article is available as a PDF (279.8 KB).

Selected References

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

  1. Algaier J., Uyeda K. Molecular cloning, sequence analysis, and expression of a human liver cDNA coding for fructose-6-P,2-kinase:fructose-2,6-bisphosphatase. Biochem Biophys Res Commun. 1988 May 31;153(1):328–333. doi: 10.1016/s0006-291x(88)81226-9. [DOI] [PubMed] [Google Scholar]
  2. Bensadoun A., Weinstein D. Assay of proteins in the presence of interfering materials. Anal Biochem. 1976 Jan;70(1):241–250. doi: 10.1016/s0003-2697(76)80064-4. [DOI] [PubMed] [Google Scholar]
  3. Bertrand L., Vertommen D., Depiereux E., Hue L., Rider M. H., Feytmans E. Modelling the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase on adenylate kinase. Biochem J. 1997 Feb 1;321(Pt 3):615–621. doi: 10.1042/bj3210615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bertrand L., Vertommen D., Feytmans E., Di Pietro A., Rider M. H., Hue L. Mutagenesis of charged residues in a conserved sequence in the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Biochem J. 1997 Feb 1;321(Pt 3):609–614. doi: 10.1042/bj3210609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Crepin K. M., Darville M. I., Hue L., Rousseau G. G. Characterization of distinct mRNAs coding for putative isozymes of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Eur J Biochem. 1989 Aug 1;183(2):433–440. doi: 10.1111/j.1432-1033.1989.tb14946.x. [DOI] [PubMed] [Google Scholar]
  6. Crepin K. M., Vertommen D., Dom G., Hue L., Rider M. H. Rat muscle 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Study of the kinase domain by site-directed mutagenesis. J Biol Chem. 1993 Jul 15;268(20):15277–15284. [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. 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]
  10. 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]
  11. Kretschmer M., Hofmann E. Inhibition of rat liver phosphofructokinase-2 by phosphoenolpyruvate and ADP. Biochem Biophys Res Commun. 1984 Nov 14;124(3):793–796. doi: 10.1016/0006-291x(84)91027-1. [DOI] [PubMed] [Google Scholar]
  12. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  13. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Lee Y. H., Ogata C., Pflugrath J. W., Levitt D. G., Sarma R., Banaszak L. J., Pilkis S. J. Crystal structure of the rat liver fructose-2,6-bisphosphatase based on selenomethionine multiwavelength anomalous dispersion phases. Biochemistry. 1996 May 14;35(19):6010–6019. doi: 10.1021/bi9600613. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Li L., Lin K., Kurland I. J., Correia J. J., Pilkis S. J. Site-directed mutagenesis in rat liver 6-phosphofructo-2-kinase. Mutation at the fructose 6-phosphate binding site affects phosphate activation. J Biol Chem. 1992 Mar 5;267(7):4386–4393. [PubMed] [Google Scholar]
  18. Müller C. W., Schulz G. E. Structure of the complex between adenylate kinase from Escherichia coli and the inhibitor Ap5A refined at 1.9 A resolution. A model for a catalytic transition state. J Mol Biol. 1992 Mar 5;224(1):159–177. doi: 10.1016/0022-2836(92)90582-5. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Pilkis S. J., Claus T. H., Kurland I. J., Lange A. J. 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase: a metabolic signaling enzyme. Annu Rev Biochem. 1995;64:799–835. doi: 10.1146/annurev.bi.64.070195.004055. [DOI] [PubMed] [Google Scholar]
  21. Rider M. H., Crepin K. M., De Cloedt M., Bertrand L., Hue L. Site-directed mutagenesis of rat muscle 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: role of Asp-130 in the 2-kinase domain. Biochem J. 1994 May 15;300(Pt 1):111–115. doi: 10.1042/bj3000111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rider M. H., Crepin K. M., De Cloedt M., Bertrand L., Vertommen D., Hue L. Study of the roles of Arg-104 and Arg-225 in the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by site-directed mutagenesis. Biochem J. 1995 Jul 1;309(Pt 1):341–346. doi: 10.1042/bj3090341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sakai A., Watanabe F., Furuya E. Cloning of cDNAs for fructose 6-phosphate 2-kinase/fructose 2,6-bisphosphatase from frog skeletal muscle and liver, and their expression in skeletal muscle. Biochem Biophys Res Commun. 1994 Feb 15;198(3):1099–1106. doi: 10.1006/bbrc.1994.1156. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Vertommen D., Bertrand L., Sontag B., Di Pietro A., Louckx M. P., Vidal H., Hue L., Rider M. H. The ATP-binding site in the 2-kinase domain of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Study of the role of Lys-54 and Thr-55 by site-directed mutagenesis. J Biol Chem. 1996 Jul 26;271(30):17875–17880. doi: 10.1074/jbc.271.30.17875. [DOI] [PubMed] [Google Scholar]
  28. Watanabe F., Sakai A., Furuya E., Uyeda K. Molecular cloning and tissue specific expression of fructose 6-phosphate,2-kinase:fructose 2,6-bisphosphatase of rat brain. Biochem Biophys Res Commun. 1994 Jan 14;198(1):335–340. doi: 10.1006/bbrc.1994.1047. [DOI] [PubMed] [Google Scholar]
  29. el-Maghrabi M. R., Pate T. M., D'Angelo G., Correia J. J., Lively M. O., Pilkis S. J. Rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Identification of essential sulfhydryl residues in the primary sequence of the enzyme. J Biol Chem. 1987 Aug 25;262(24):11714–11720. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES