Site Directed Mutagenesis as a Tool to Study Enzyme Catalysis

  • John R. Coggins
Part of the Progress in Mathematics book series (NSSA)


Although the use of site-directed mutagenesis to engineer proteins, which was introduced in the preceding chapter, is less than 10 years old there have already been many publications describing the application of this technique to the study of enzymes. In this chapter I shall give some examples to illustrate how enzymologists are using site-diected mutagenesis to study enzyme catalysis and at the same time discuss the advantages and the limitations of the technique. Further examples may be found in the several excellent reviews that have appeared recently (Blow et al., 1986; Fersht et al., 1986a; Leatherbarrow & Fersht, 1986; 1987; Oxender & Fox, 1987; Shaw, 1987; Wells and Estell, 1988).


Serine Protease Mutant Enzyme Amino Acid Side Chain Protein Engineer Preceding Chapter 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Blow, D.M., Fersht, A.R. and Winter, G., 1986, “Design, Construction and Properties of Novel Protein Molecules”, Royal Society, London.Google Scholar
  2. Clarke, A.R., Atkinson, T. and Holbrook, J.J., 1989, From analysis to synthesis: new ligand binding sites on the lactate dehydrogenase framework, Trends. Biochem. Sci. 14: 101.PubMedCrossRefGoogle Scholar
  3. Craik, C.S., Largman, C., Fletcher, T., Roczniak, S., Barr, P.J., Fletterick, R. and Rutter, W.J., 1985, Redesigning trypsin: alteration of the substrate specificity, Science 228: 291.PubMedCrossRefGoogle Scholar
  4. Fersht, A.R., 1985, “Enzyme Structure and Mechanism” ( 2nd ed. ), Freeman, San Francisco.Google Scholar
  5. Fersht, A.R., 1987, Kinetic aspects of purposely modified proteins, in “Protein Engineering”, Oxender, D.L. and Fox, C.F. (eds.), Liss, New York p. 225.Google Scholar
  6. Fersht, A.R. and Leatherbarrow, R.J., 1987, Structure and activity of tyrosyl-t-RNA synthetase, in “Protein Engineering”, Oxender, D.L. and Fox, C.F. (eds.), Liss, New York p. 269.Google Scholar
  7. Fersht, A.R., Leatherbarrow, R.J. and Wells, T.N.C., 1986a, Binding energy and catalysis: a lesson from protein engineering of tyrosyl-tRNA synthetase, Trends. Biochem. Sci. 11: 321.Google Scholar
  8. Fersht, A.R., Leatherbarrow, R.J. and Wells, T.N.C., 1986b, Structure and activity of tyrosyl-tRNA synthetase: the hydrogen bond in catalysis and specificity, Phil. Trans. R. Soc. Lond. A 317: 305.Google Scholar
  9. Gardell, S.J., Craik, C.S., Hilvert, D., Urdea, M.S. and Rutter, W.J., 1985, Site-directed mutagenesis shows that tyrosine-248 of carboxypeptidase A does not play a crucial role in catalysis, Nature 317: 551.Google Scholar
  10. Graf, L., Craik, C.S., Patthy, A., Roczniak, S., Fletterick, R.J. and Rutter, W.J., 1987, Selective alteration of substrate specificity by replacement of aspartic acid-189 with lysine in the binding pocket of trypsin, Biochemistry 26: 2616.PubMedCrossRefGoogle Scholar
  11. Hartsuck, J.A. and Lipscomb, W.N., 1971, Carboxypeptidase A, in “The Enzymes”, 3rd edn., Volume III, Boyer, P.D. (ed.), Academic Press, New York p. 1.Google Scholar
  12. Karplus, M., 1987, The prediction and analysis of mutant structures, in “Protein Engineering”, Oxender, D.L. and Fox, C.F. (eds.), Liss, New York p. 35.Google Scholar
  13. Leatherbarrow, R.J. and Fersht, A.R., 1986, Protein Engineering 1: 7.PubMedCrossRefGoogle Scholar
  14. Leatherbarrow, R.J., Fersht, A.R. and Winter, G., 1985, Transition-state stabilisation in the mechanism of tyrosyl-tRNA synthetase revealed by protein engineering, Proc. Natl. Acad. Sci. USA 82: 7840.Google Scholar
  15. Leatherbarrow, R.J. and Fersht, A.R., 1987, Use of protein engineering to study enzyme mechanisms’, in “Enzyme Mechanisms”, Page, M.I. and Williams, A. (eds), The Royal Society of Chemistry, London, p. 78.Google Scholar
  16. Lipscomb, W.N., 1980, Carboxypeptidase A mechanisms, Proc. Natl. Acad. Sci. USA 77: 3875.Google Scholar
  17. Markley, J.L., 1987, One-and two-dimensional nmr spectroscopic investigations of the consequences of amino acid replacements in proteins, in “Protein Engineering”, Oxender, D.L. and Fox, C.F. (eds.), Liss, New York p. 15.Google Scholar
  18. Monteilhet, C., Blow, D.M. and Brick, P., 1984, Interaction of crystalline tyrosyl-tRNA synthetase with adenosine, adenosine monophosphate, adenosine triphosphate and pyrophosphate in the presence of tyrosinol, J. Mol. Biol. 173: 477.Google Scholar
  19. Oxender, D.L. and Fox, C.F., 1987, “Protein Engineering”, Liss, New York.Google Scholar
  20. Pauling, L., 1948, Nature of forces between large molecules of biological interest, Nature 161: 707.PubMedCrossRefGoogle Scholar
  21. Robertus, J.D., Kraut, J., Alden, R.A. and Birktoft, J.J., 1972, Subtilisin: a stereochemical mechanism involving transition-state stabilisation, Biochemistry 11: 4293.PubMedCrossRefGoogle Scholar
  22. Rossi, J. and Zoller, M., 1987, Site-specific and regionally directed mutagenesis of protein-encoding sequences, in “Protein Engineering”, Oxender, D.L. and Fox, C.F. (eds.), Liss, New York p. 51.Google Scholar
  23. Rubin, J. and Blow, D.M., 1981, Amino acid activation in crystalline tyrosyl-t-RNA synthetase from Bacillus stearothermophilus, J. Mol. Biol. 145: 489.Google Scholar
  24. Russell, A.J. and Fersht, A.R., 1987, Rational modification of enzyme catalysis by engineering surface charge, Nature 328: 496.PubMedCrossRefGoogle Scholar
  25. Rutter, W.J., Gardell, S.J., Rocniak, S., Hilvert, D., Sprang, S., Fletterick, R.J. and Craik, C.S., 1987, Redesigning proteins by genetic engineering, in “Protein Engineering”, Oxender, D.L. and Fox, C.F. (eds.), Liss, New York p. 257.Google Scholar
  26. Shaw, W.V., 1987, Protein Engineering: the design, synthesis and characterisation of factitious proteins’ Biochem. J. 246: 1.Google Scholar
  27. Sprang, S., Standing, T., Fletterick, R.J., Stroud, R.M., Finer-Moore, J., Xuong, N-H, Hamlin, R., Rutter, W.J. and Craik, C.S., 1987, The three-diMensional structure of Asn102 mutant of trypsin: role of Asp102 in serine protease catalysis, Science 237: 905.PubMedCrossRefGoogle Scholar
  28. Stroud, R.M., Krieger, M., Koeppe, R.E., Kossiakoff, A.A. and Chambers, J.L., 1975, Structure function relationships in the serine proteases, in “Proteases and Biological Control”, Reich, E., Rifkin, D.B. and Shaw, E., eds., Cold Spring Harbor Laboratory, New York, p. 13.Google Scholar
  29. Thomas, P.G., Russell, A.J. and Fersht, A.R., 1985, Tailoring the pH-dependence of enzyme catalysis using protein engineering, Nature 318: 375.CrossRefGoogle Scholar
  30. Walsh, C., 1979, “Enzymatic Reaction Mechanisms”, Freeman, San Francisco.Google Scholar
  31. Wells, J.A., Cunningham, B.C., Graycar, T.P. and Estell, D.A., 1986, Importance of hydrogen-bond formation in stabilizing the transition state of subtilisin, Phil. Trans. R. Soc. Lond. A317: 415.Google Scholar
  32. Wells, J.A. and Estell, D.A., 1988, Subtilisin - an enzyme designed to be engineered, Trends. Biochem. Sci. 13: 291.PubMedCrossRefGoogle Scholar
  33. Wells, T.N.C. and Fersht, A.R., 1985, Hydrogen bonding in enzymatic catalysis analysed by protein engineering, Nature 316: 656.CrossRefGoogle Scholar
  34. Winter, G., Fersht, A.R., Wilkinson, A.J., Zoller, M. and Smith, M., 1982, Redesigning enzyme structure by site-directed mutagenesis: tyrosyl tRNA synthetase and ATP binding, Nature 299: 756.PubMedCrossRefGoogle Scholar
  35. Wolfenden, R. and Frick, L., 1987, Transition state affinity and the design of enzyme inhibitors, in “Enzyme Mechanisms”, Page, M.I. and Williams, A. (eds), The Royal Society of Chemistry, London, p. 97.Google Scholar

Copyright information

© Springer Science+Business Media New York 1989

Authors and Affiliations

  • John R. Coggins
    • 1
  1. 1.Department of BiochemistryUniversity of GlasgowGlasgowScotland

Personalised recommendations