Abstract
The dimeric enzyme CTP:glycerol-3-phosphate cytidylyltransferase (GCT) displays strong negative cooperativity between the first and second binding of its substrate, CTP. Using NMR to study the allosteric mechanism of this enzyme, we observe widespread chemical shift changes for the individual CTP binding steps. Mapping these changes onto the molecular structure allowed the formulation of a detailed model of allosteric conformational change. Upon the second step of ligand binding, NMR experiments indicate an extensive loss of conformational exchange broadening of the backbone resonances of GCT. This suggests that a fraction of the free energy of negative cooperativity is entropic in origin.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on SpringerLink
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Henis, Y.I., & Levitzki, A., in Allosteric enzymes (ed. Hervé, G.) 154–174 (CRC Press, Inc, Boca Raton, Florida; 1989)
Pooley, H.M., Abellan, F. X. & Karamata, D. J. Gen. Microbiol. 137, 921–928 (1991).
Park, Y.S., Sweitzer, T.D., Dixon, J.E. & Kent, C. J. Biol. Chem. 268, 16648–16654 (1993).
Sanker, S., Campbell, H. & Kent, C. J. Biol. Chem. In the press. (2001).
Weber, C.H., Park, Y.S., Sanker, S., Kent, C. & Ludwig, M.L. Structure 7, 1113–1124 (1999).
Park, Y.S. et al. J. Biol. Chem. 272, 15161–15166 (1997).
Pellecchia, M., Sebbel, P., Hermanns, U., Wüthrich, K. & Glockshuber, R. Nature Struct. Biol. 6, 336–339 (1999).
Koshland, D.E. Jr., Némethy, G. & Filmer, D. Biochemistry 5, 365–385 (1996).
Seydoux, F., Malhotra, O.P. & Bernhard, S.A. CRC Crit. Rev. Biochem. 2, 227–236 (1974).
Biesecker, G., Harris, J.I., Thierry, J.C., Walker, J.E. & Wonacott, A.J. Nature 266, 328–333 (1977).
Leslie, A.G.W. & Wonacott, A.J. J. Mol. Biol. 165, 375–391 (1983).
Leslie, A.G.W. & Wonacott, A.J. J. Mol. Biol. 178, 743–772 (1984).
Finer-Moore, J. et al. J. Mol. Biol. 232, 1101–1116 (1993).
Anderson, A.C., O'Neil, R.H., DeLano, W.L. & Stroud, R.M. Biochemistry 38, 13829–13836 (1999).
Koshland, D.E. Jr., Curr. Opin. Struct. Biol. 6, 757–761 (1996).
Biemann, H.-P. & Koshland, D.E. Jr., Biochemistry 33, 629–634 (1994).
Orekov, V.Y., Pervushin, K.V. & Arseniev, A.S. Eur. J. Biochem. 219, 887–896 (1994).
Akke, M. & Palmer, A.G. J. Am. Chem. Soc. 118, 911–912 (1996).
Wang, L. et al. Proc. Natl. Acad. Sci. USA 98, 7684–7689 (2001).
Lee, A.L., Kinnear, S.A. & Wand, A.J. Nature Struct. Biol. 7, 72–77 (2000).
Loh, A.P., Pawley, N., Nicholson, L.K. & Oswald, R.E. Biochemistry 40, 4590–4600 (2001).
Mäler, L., Blankenship, J., Rance, M. & Chazin, W. J. Nature Struct. Biol. 7, 245–250 (2000).
Hamazaki, T., Lee, W., Arrowsmith, C.H., Muhandiram, D.R. & Kay, L.E. J. Am. Chem. Soc. 116, 11655–11666 (1994).
Boucher, W., Laue, E.D., Campbell-Burke, S. & Domaille, P.J. J. Biomol. NMR 2, 631–637 (1992).
Shortle, D. J. Magn. Reson. Ser. B 105, 88–90 (1994).
Bartels, C., Xia, T.-H., Billeter, M., Güntert, P. & Wüthrich, K. J. Biomol. NMR 5, 1–10 (1995).
Delaglio, F. et al. J. Biomol. NMR 6, 277–293 (1995).
Johnson, B.A. & Blevins, R.A. J. Biomol. NMR 4, 603–614 (1994).
Koradi, R., Billeter, M. & Wüthrich, K. J. Mol. Graph. 14, 51–55 (1996).
Segel, I.H., Enzyme kinetics (John Wiley & Sons, Inc., New York; 1975).
Acknowledgements
This work was supported by grants from the National Institutes of Health. We thank C. Wilde for developing the protein purification procedure, and C. Weber and M.L. Ludwig for guiding us through the crystal structure and for stimulating discussions in general. We thank G. Yip for his contributions to the NMR relaxation experiments. The Keck Foundation, NIH and NSF are acknowledged for support of the 800 MHz system.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Stevens, S., Sanker, S., Kent, C. et al. Delineation of the allosteric mechanism of a cytidylyltransferase exhibiting negative cooperativity. Nat Struct Mol Biol 8, 947–952 (2001). https://doi.org/10.1038/nsb1101-947
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1038/nsb1101-947
This article is cited by
-
New Insights into Active Site Conformation Dynamics of E. coli PNP Revealed by Combined H/D Exchange Approach and Molecular Dynamics Simulations
Journal of the American Society for Mass Spectrometry (2016)
-
Accurate determination of rates from non-uniformly sampled relaxation data
Journal of Biomolecular NMR (2016)
-
The role of protein dynamics in allosteric effects—introduction
Biophysical Reviews (2015)
-
Millisecond dynamics in the allosteric enzyme imidazole glycerol phosphate synthase (IGPS) from Thermotoga maritima
Journal of Biomolecular NMR (2009)
-
Self-consistent residual dipolar coupling based model-free analysis for the robust determination of nanosecond to microsecond protein dynamics
Journal of Biomolecular NMR (2008)