Abstract
The enzyme-catalysed transfer of a phosphoryl group from ATP is an important reaction in a wide variety of biological processes. We demonstrate here the essential function of an aspartate group in the catalysis of phosphoryl transfer by Escherichia coli phosphofructokinase, and the minor role of an arginine residue. We have used oligonucleotide-directed mutagenesis to replace two amino-acid residues which X-ray analysis has shown to be close to the transferred phosphoryl group and we have analysed the forward and back reactions of the mutant enzymes by steady-state kinetics. Changing Asp 127 to Ser reduced the turnover number by a factor of 18,000 in the forward direction and 3,100 in the back reaction, and the Michaelis constant for fructose 1,6-bisphosphate in the reverse reaction by a factor of 45. This shows that this aspartate is a key residue in the rate enhancement by the enzyme, probably acting as a base in the reaction mechanism, and that it also destabilizes the product complex. Changing Arg 171 to Ser reduced the turnover numbers by about 3.4, showing that this arginine has only a minor effect on the catalysis.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
- Purchase on Springer Link
- 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
Morrison, J. F. & Heyde, E. A. Rev. Biochem. 41, 29–54 (1972).
Benkovic, S. J. & Schray, K. J. Enzymes 8, 201–238 (1971).
Knowles, J. R. A. Rev. Biochem. 49, 877–918 (1980).
Buchwald, S. L., Hansen, D. E., Hassett, A. & Knowles, J. R. Meth. Enzym. 87, 279–301 (1982).
Uyeda, K. Adv. Enzym. 48, 193–244 (1979).
Hers, H. G. & van Schafteningen, E. Biochem. J. 206, 1–12 (1982).
Evans, P. R. & Hudson, P. J. Nature 279, 500–504 (1979).
Evans, P. R., Farrants, G. W. & Hudson, P. J. Phil. Trans. R. Soc. B293, 53–62 (1981).
Hellinga, H. W. & Evans, P. R. Eur. J. Biochem. 149, 363–373 (1985).
Poorman, R. A., Randolph, A., Kemp, R. G. & Heinrikson, R. L. Nature 309, 467–469 (1984).
Jarvest, R. L., Lowe, G. & Potter, B. V. L. Biochem. J. 199, 427–432 (1981).
Smith, M. A. Rev. Genet. 19, 423–462 (1985).
Viola, R. E. & Cleland, W. W. Biochemistry 17, 4111–4117 (1978).
Anderson, C. M., Stenkamp, R. E., McDonald, R. C. & Steitz, T. A. J. molec. Biol. 123, 207–219 (1978).
Bromilow, R. H. & Kirby, A. J. J. chem. Soc. Perkin Trans. II, 149–155 (1972).
Cotton, F. A., Hazen, E. E. & Legg, M. J. Proc. natn. Acad. Sci. U.S.A. 76, 2551–2555 (1979).
Springs, B. & Haake, P. Tetrahedron Lett. 37, 3223–3226 (1977).
Cotton, F. A., Cour, T., Hazen, E. E. & Legg, M. J. Biochim. biophys. Acta 481, 1–5 (1977).
Segel, I. H. Enzyme Kinetics (Wiley, New York, 1974).
Blangy, D., Buc, H. & Monod, J. J. molec. Biol. 31, 13–35 (1968).
Kotlarz, D. & Buc, H. Biochim. biophys. Acta 484, 35–48 (1977).
Zoller, M. J. & Smith, M. Meth. Enzym. 100, 468–500 (1983).
Carter, P. J., Bedouelle, H. & Winter, G. Nucleic Acids Res. 13, 4431–4443 (1985).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Hellinga, H., Evans, P. Mutations in the active site of Escherichia coli phosphofructokinase. Nature 327, 437–439 (1987). https://doi.org/10.1038/327437a0
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1038/327437a0
This article is cited by
-
Metabolite profiling and associated gene expression reveal two metabolic shifts during the seed-to-seedling transition in Arabidopsis thaliana
Plant Molecular Biology (2017)
-
Evolution of the Genetic Code by Incorporation of Amino Acids that Improved or Changed Protein Function
Journal of Molecular Evolution (2013)
-
Chemistry: Designer debacle
Nature (2008)
-
Three-dimensional structure of the ATPase fragment of a 70K heat-shock cognate protein
Nature (1990)
-
Active-site mutants altering the cooperativity of E. coliphosphofructokinase
Nature (1990)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.
