Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Substrate-assisted catalysis as a mechanism for GTP hydrolysis of p21ras and other GTP-binding proteins

Nature Structural Biology volume 2, pages 36–44 (1995)Cite this article

Abstract

Despite many advances in understanding the structure and function of GTP-binding proteins the mechanism by which these molecules switch from the GTP-bound on-state to the GDP-bound off-state is still poorly understood. Theoretical studies suggest that the activation of the nucleophilic water which hydrolyzes GTP needs a general base. Such a base could not be located in any of the many GTP-binding proteins. Here we present a unique type of linear free energy relationships that not only supports a mechanism for p21rasin which the substrate GTP itself acts as the catalytic base driving the GTPase reaction but can also help to explain why certain mutants of p21ras are oncogenic and others are not.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Similar content being viewed by others

References

  1. Bourne, H.R., Sanders, D.W. & McCormick, F. GTPase superfamily: a conserved switch for diverse cell functions. Nature 348, 125–132 (1990).

    Article  CAS  PubMed  Google Scholar 

  2. Lowy, D.R. & Willumsen, B.M. Function and regulation of Ras. A. Rev. Biochem. 62, 851–891 (1993).

    Article  CAS  Google Scholar 

  3. Zhang, X.-f. et al. Normal and oncogenic p21 ras proteins bind to the amino-terminal regulatory domain of c-Raf-1. Nature 364, 308–313 (1993).

    Article  CAS  PubMed  Google Scholar 

  4. Warne, P.H., Rodriguez-Viciana, P. & Downward, J. Direct interaction of Ras and the amino-terminal region of Raf-1 in vitro. Nature 364, 352–355 (1993).

    Article  CAS  PubMed  Google Scholar 

  5. van Aelst, L., Barr, M., Marcus, S., Polverino, A. & Wigler, M. Complex formation between RAS and RAF and other protein kinases. Proc. natn. Acad. Sci. U.S.A. 90, 6213–6219 (1993).

    Article  CAS  Google Scholar 

  6. Vojtek, A.B., Hollenberg, S.M. & Cooper, J.A., Ras interacts directly with the serine/threonine kinase Raf. Cell 74, 205–214 (1993).

    Article  CAS  PubMed  Google Scholar 

  7. Boguski, M.S. & McCormick, F. Proteins regulating Ras and its relatives. Nature 366, 643–654 (1993)

    Article  CAS  PubMed  Google Scholar 

  8. Der, D., Finkel, T. & Cooper, G.M. Biological and biochemical properties of human rasH genes mutated at codon 61. Cell 44, 167–176 (1986).

    Article  CAS  PubMed  Google Scholar 

  9. Seeburg, P.H., Colby, W.W., Capon, D.J., Goedel, D.V. & Levinson, A.D. Biological properties of human c-Ha-ras 1 genes mutated at codon 12. Nature 312, 71–75 (1984).

    Article  CAS  PubMed  Google Scholar 

  10. Pai, E.F. et al. Structure of the guanine-nucleotide-binding domain of the Ha-ras oncogene product p21 in the triphosphate conformation. Nature 341, 209–214 (1989).

    Article  CAS  PubMed  Google Scholar 

  11. Milburn, M.V. et al. Molecular switch for signal transduction: structural differences between active and inactive forms of protooncogenic ras proteins. Science 247, 939–945 (1990).

    Article  CAS  PubMed  Google Scholar 

  12. Pai, E.F. et al. Refined crystal structure of the triphosphate conformation of H-ras p21 at 1.35 Å resolution: implications for the mechanism of GTP hydrolysis. EMBO J. 9, 2351–2359 (1990).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Privé, G.G. et al. X-ray crystal structures of transforming p21 ras mutants suggest a transition-state stabilization mechanism for GTP hydrolysis. Proc. natn. Acad. Sci. U.S.A. 80, 3649–3653 (1992).

    Article  Google Scholar 

  14. Toney, M.D. & Kirsch, J.F. Brønsted analysis of the restoration of activity to a mutant enzyme by exogenous amines. Science 243, 1485–1488 (1989).

    Article  CAS  PubMed  Google Scholar 

  15. Fersht, A.R., Leatherbarrow, R.J. & Wells, T.N.C. Quantitative analysis of structure-activity relationships in engineered proteins by linear free-energy relationships. Nature 322, 284–286 (1986).

    Article  CAS  Google Scholar 

  16. Warshel, A. Computer Modeling of Chemical Reactions in Enzymes and Solutions. (John Wiley, New York, 1991).

  17. Warshel, A., Schweins, T. & Fothergill, M. Linear free energy relationships in enzymes. Theoretical analysis of the reaction of tyrosyl-tRNA synthetase. J. Am. chem. Soc. 116, 8437–8442 (1994).

    Article  CAS  Google Scholar 

  18. Freeh, M. et al. Role of Glutamine-61 in the hydrolysis of GTP by p21 H-ras: An experimental and theoretical study. Biochemistry 33, 3237–3244 (1994).

    Article  Google Scholar 

  19. Chung, H.H., Benson, D.R. & Schultz, P.G. Probing the structure and mechanism of ras protein with an expanded genetic code. Science 259, 806–809 (1992).

    Article  Google Scholar 

  20. Langen, R., Schweins, T. & Warshel, A. On the mechanism of guanosine triphosphate hydrolysis in ras p21 proteins. Biochemistry 31, 8691–8696 (1992).

    Article  CAS  PubMed  Google Scholar 

  21. Calès, C., Hancock, J.F., Marshall, C.J. & Hall, A. The cytoplasmic protein GAP is implicated as the target for regulation by the ras gene product. Nature 332, 548–551 (1988).

    Article  PubMed  Google Scholar 

  22. John, J. et al. Kinetic and structural analysis of the Mg2+-binding site of the guanine nucleotide-binding protein p21H-ras. J. biol. Chem. 268, 923–929 (1993).

    CAS  PubMed  Google Scholar 

  23. Noel, J.P., Hamm, H.E. & Sigler, P.B. The 2.2 Å crystal structure of transducin-α complexed with GTPψS. Nature 366, 654–663 (1993).

    Article  CAS  PubMed  Google Scholar 

  24. Krengel, U. et al. Three-dimensional structures of H-ras p21 mutants: molecular basis for their inability to function as signal switch molecules. Cell 62, 539–548 (1990).

    Article  CAS  PubMed  Google Scholar 

  25. Wittinghofer, A., Pai, E.F. & Goody, R.S. in GTPases in Biology I (eds Dickey, B.F. & Birnbauer, L.) 195–211 (Springer-Verlag, Berlin-Heidelberg-New York, 1993).

    Book  Google Scholar 

  26. Gideon, P. et al. Mutational and kinetic analysis of the GTPase-activating protein (GAP)-p21 interaction: The C-terminal domain of GAP is not sufficient for full activity. Molec. Cell Biol. 12, 2050–2056 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Fasano, O. et al. Analysis of the transforming potential of the human H-ras gene by random mutagenesis. Proc. natn. Acad. Sci. U.S.A. 81, 4008–4012 (1984).

    Article  CAS  Google Scholar 

  28. Kleuss, C., Raw, A.S., Lee, E., Sprang, S.R. & Gilman, A.G. Mechanism of GTP hydrolysis by G-protein α subunits. Proc. natn. Acad. Sci. U.S.A. 91, 9828–9831 (1994).

    Article  CAS  Google Scholar 

  29. Schweins, T., Langen, R. & Warshel, A. Why have mutagenesis studies not located the general base in ras p21? Nature Struct. Biol. 1, 476–484 (1994).

    Article  CAS  PubMed  Google Scholar 

  30. Perona, J.J., Rould, M.A. & Steitz, T.A. Structural basis for transfer RNA aminoacylation by Escherichia coli glutaminyl-tRNA synthetase. Biochemistry 32, 8758–8771 (1993).

    Article  CAS  PubMed  Google Scholar 

  31. Cavarelli, J. et al. The active site of yeast aspartyl-tRNA synthetase: structural and functional aspects of the aminoacylation reaction. EMBO J. 13, 327–337 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Gouaux, J.E., Krause, K.L. & Lipscomb, W.N. The catalytic mechanism of Escherichia coli aspartate carbamoyltransferase: a molecular modelling study. Biochem. biophys. Res. Comm. 142, 893–897 (1987).

    Article  CAS  PubMed  Google Scholar 

  33. Jeltsch, A., Alves, J., Woifes, H., Maass, G. & Pingoud, A. Substrate-assisted catalysis in the cleavage of DNA by the EcoRI and EcoRV restriction enzymes. Proc. natn. Acad. Sci. U.S.A. 90, 8499–8503 (1993).

    Article  CAS  Google Scholar 

  34. Feuerstein, J., Goody, R.S. & Webb, M.R. The mechanism of guanosine nucleotide hydrolysis by p21 c-Ha-ras. J. biol. Chem. 264, 6188–6190 (1989).

    CAS  PubMed  Google Scholar 

  35. Schlichting, I. et al. Time-resolved X-ray crystallographic study of the conformational change in Ha-ras p21 protein on GTP hydrolysis. Nature 345, 309–315 (1990).

    Article  CAS  PubMed  Google Scholar 

  36. Herrmann, C., Martin, G.A. & Wittinghofer, A. Quantitative analysis of the complex between p21ras and the Ras-binding domain of the human Raf-1 protein kinase. J. Biol. Chem. (in the press).

  37. Mistou, M.-Y. et al. Mutations of Ha-ras p21, that define important regions for the molecular mechanism of the SDC25 C-domain, a guanine nucleotide dissociation stimulator. EMBO J. 11, 2391–2397 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Eccleston, J.F., Moore, K.J.M., Morgan, L., Skinner, R.H. & Lowe, P.N. Kinetics of interaction between normal and proline 12 ras and the GTPase-activating proteins, p120-GAP and neurofibromin. J. biol. Chem. 268, 27012–27019 (1993).

    CAS  PubMed  Google Scholar 

  39. Franken, S.M. et al. Three-dimensional structures and properties of a transforming and a nontransforming glycine-12 mutant of p21H-ras. Biochemistry 32, 8411–8420 (1993).

    Article  CAS  PubMed  Google Scholar 

  40. Bollag, G. & McCormick, F. Differential regulation of rasGAP and neurofibromatosis gene product activities. Nature 351, 576–579 (1991).

    Article  CAS  PubMed  Google Scholar 

  41. Bryan, P., Pantoliano, M.W., Quill, S.G., Hsiao, H.-Y. & Poulos, T. Site-directed mutagenesis and the role of the oxyanion hole in subtilisin. Proc. natn. Acad. Sci. U.S.A. 83, 3743–3745 (1986).

    Article  CAS  Google Scholar 

  42. Warshel, A., Sussman, F. & Hwang, J.-K. Evaluation of catalytic free energies in genetically modified proteins. J. molec. Biol. 201, 139–159 (1988).

    Article  CAS  PubMed  Google Scholar 

  43. Menard, R. et al. Contribution of the glutamine 19 side chain to transition-state stabilization in the oxyanion hole of papain. Biochemistry 30, 8924–8928 (1991).

    Article  CAS  PubMed  Google Scholar 

  44. Coleman, D.E. et al. Structures of active conformations of Giα1 and the mechanism of GTP hydrolysis. Science 265, 1405–1412 (1994).

    Article  CAS  PubMed  Google Scholar 

  45. Sondek, J., Lambright, D.G., Noel, J.P., Hamm, H.E. & Sigler, P.B. GTPase mechanism of G proteins from the 1.7-Å crystal structure of transducin α•GDP•AlF4−. Nature 372, 276–279 (1994).

    Article  CAS  PubMed  Google Scholar 

  46. Chabre, M. Aluminofluoride and beryllofluoride complexes: new phosphate analogs in enzymology. Trends biol. Sci. 15, 6–10 (1990).

    Article  CAS  Google Scholar 

  47. Gupta, S.K. et al. Analysis of the fibroblast transformation potential of GTPase-deficient gip2 oncogenes. Molec. Cell Biol. 12, 190–197 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Landis, C.A. et al. GTPase inhibiting mutations activate the chain of GS and stimulate adenylyl cyclase in human pituitary tumours. Nature 340, 692–696 (1989).

    Article  CAS  PubMed  Google Scholar 

  49. van Dop, C., Tsubokawa, M., Bourne, H.R. & Ramachandran, J. Amino acid sequence of retinal transducin at the site ADP-ribosylated by cholera toxin. J. biol. Chem. 259, 696–698 (1984).

    CAS  PubMed  Google Scholar 

  50. Skinner, R.H. et al. Use of the Glu-Glu-Phe C-terminal epitope for rapid purification of the catalytic domain of normal and mutant ras GTPase-activating proteins. J. biol. Chem. 266, 14163–14166 (1991).

    CAS  PubMed  Google Scholar 

  51. Müller, C.W. & Schulz, G.E. Structure of the complex between adenylate kinase from Escherichia coli and the inhibitor Ap5A refined at 1.9 Å resolution. J. molec. Biol. 224, 159–177 (1992).

    Article  PubMed  Google Scholar 

  52. Berchtold, H. et al. Crystal structure of active elongation factor Tu reveals major domain rearrangements. Nature 365, 126–132 (1993).

    Article  CAS  PubMed  Google Scholar 

  53. Kjeldgaard, M., Nissen, P., Thirup, S. & Nyborg, J. The crystal structure of elongation factor EF-Tu from Thermus aquaticus in the GTP conformation. Structure 1, 35–50 (1993).

    Article  CAS  PubMed  Google Scholar 

  54. Cool, R.H. & Parmeggiani, A. Substitution of histidine-84 and the GTPase mechanism of elongation factor Tu. Biochemistry 30, 362–366 (1991).

    Article  CAS  PubMed  Google Scholar 

  55. Scherer, A. et al. Crystallization and preliminary X-ray analysis of the human c-H-ras oncogene product p21 complexed with GTP analogues. J. molec. Biol. 206, 257–259 (1989).

    Article  CAS  PubMed  Google Scholar 

  56. Duggleby, R.G. & Clarke, R.B. Experimental designs for estimating the parameters of the Michaelis-Menten equation from progress curves of enzyme-catalyzed reactions. Bioch. et Biophys. Acta. 1080, 231–236 (1991).

    CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Max-Planck-lnstitut für molekulare Physiologie, Abteilung Strukturelle Biologie, Rheinlanddamm 201, 44139, Dortmund, Germany

    Thomas Schweins & Alfred Wittinghofer

  2. Max-Planck-lnstitut für Medizinische Forschung, Jahnstr. 29, 69120, Heidelberg, Germany

    Matthias Geyer, Klaus Scheffzek & Hans Robert Kalbitzer

  3. Department of Chemistry, University of Southern California, Los Angeles, California, 90089-1062, USA

    Arieh Warshel

Authors
  1. Thomas Schweins
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Matthias Geyer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Klaus Scheffzek
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Arieh Warshel
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hans Robert Kalbitzer
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Alfred Wittinghofer
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Schweins, T., Geyer, M., Scheffzek, K. et al. Substrate-assisted catalysis as a mechanism for GTP hydrolysis of p21ras and other GTP-binding proteins. Nat Struct Mol Biol 2, 36–44 (1995). https://doi.org/10.1038/nsb0195-36

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsb0195-36

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing