Altering the Structure of Enzymes by Site-Directed Mutagenesis

  • W. H. J. Ward
  • A. R. Fersht


Site-directed mutagenesis of enzymes allows allows the direct study of the contributions of specific side-chains to substrate binding and catalysis. This has led to a breakthrough in understanding relationships between structure and function. Tyrosyl-tRNA synthetase (TyrTS) from Bacillus stearothermophilus has been a paradigm for protein engineering studies on structure reactivity relationships in catalysis. Kinetic analysis of TyrTS mutants has given quantitative information which can be applied to many proteins. The interaction energy between the enzyme and its substrates during catalysis has been determined, allowing measurement of the apparent strengths of hydrogen bonds and salt bridges. It has been shown directly that catalysis results from stabilization of the transition state. The gross structure of TyrTS has also been investigated. The enzyme comprises 2 subunits of identical composition, and each monomer has 2 discrete functional domains: one which binds tRNA and the other which contains the active site. tRNA interacts with both subunits, binding to one and then being charged at the other subunit of the dimer. Each monomer has a complete active site, but only 1 site in each dimer functions catalytically. TyrTS is thus a classical example of an enzyme with half-of-the-sites activity. The mechanism of TyrTS has been studied and it has been shown that each dimer uses the same active site repeatedly. The second subunit has no detectable activity so that the enzyme has long-lasting asymmetry in function. Asymmetry is an inherent property and is not induced by binding of substrate. This accounts for half-of-the-sites activity and shows that the enzyme has an asymmetrical structure in solution, contrasting with the structure in crystals which is symmetrical about the subunit interface. A monomer of the enzyme is probably too small to allow both recognition and charging of tRNA, explaining the requirement for the enzyme to function as an asymmetric dimer. The enzyme appears to bind two molecules of Tyr sequentially to the same site during charging of 1 molecule of tRNA. The second molecule of Tyr perhaps aids the dissociation of charged tRNA by displacing the tyrosyl moiety from its binding site.


Protein Engineering Mutant Enzyme Subunit Interface Biphasic Kinetic Crystalline Enzyme 
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.


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  1. 1.
    Leatherbarrow RJ, Fersht AR (1986) Protein Engineering 1:7–16PubMedCrossRefGoogle Scholar
  2. 2.
    Carter P (1986) Bioehem J 237:1–7Google Scholar
  3. 3.
    Knowles JR (1987) Science 236:1252–1258PubMedCrossRefGoogle Scholar
  4. 4.
    Shaw WV (1987) Biochem J 246:1–17PubMedGoogle Scholar
  5. 5.
    Ward WHJ (1987) Trends Biochem Sci 12:28–31CrossRefGoogle Scholar
  6. 6.
    Winter G, Fersht AR, Wilkinson AJ, Zoller M, Smith M (1982) Nature 299:756–758PubMedCrossRefGoogle Scholar
  7. 7.
    Winter G, Koch GLE, Hartley BS, Barker DG (1983) Eur J Bioehem 132:383–387CrossRefGoogle Scholar
  8. 8.
    Fersht AR, Ashford JS, Bruton CJ, Jakes R, Koch GLE, Hartley BS (1975) Biochemistry 14:1–4PubMedCrossRefGoogle Scholar
  9. 9.
    Fersht AR, Mulvey RS, Koch GLE (1975) Biochemistry 14:13–18PubMedCrossRefGoogle Scholar
  10. 10.
    Blow DM, Brick P (1985) In: Jurnak F, McPherson A (eds) Biological Macromolecules and Assemblies Nucleic Acids and Interative Proteins, Wiley, New York 2:442–469Google Scholar
  11. 11.
    Jakes R, Fersht AR (1975) Biochemistry 14:3350–3356PubMedCrossRefGoogle Scholar
  12. 12.
    Wells TNC, Ho C, Fersht AR (1986) Biochemistry 25:6603–6608PubMedCrossRefGoogle Scholar
  13. 13.
    Calendar R, Berg P (1966) Biochemistry 5:1681–1690PubMedCrossRefGoogle Scholar
  14. 14.
    Waye MMY, Winter G, Wilkinson AJ, Fersht AR (1983) EMBO J 2:1827–1830PubMedGoogle Scholar
  15. 15.
    Brick P, Blow D (1987) J Molec Biol 194:287–297PubMedCrossRefGoogle Scholar
  16. 16.
    Brown KA, Brick P, Blow DM (1987) Nature 326:416–418PubMedCrossRefGoogle Scholar
  17. 17.
    Carter P, Winter G, Wilkinson AJ, Fersht AR (1984) Cell 38:835–840PubMedCrossRefGoogle Scholar
  18. 18.
    Fersht AR, Leatherbarrow RJ, Wells TNC (1986) Nature 322:284–286CrossRefGoogle Scholar
  19. 19.
    Fersht AR, Leatherbarrow RJ, Wells TNC (1987) Biochemistry 26:6030–6038PubMedCrossRefGoogle Scholar
  20. 20.
    Fersht AR, Shi JP, Knill-Jones J, Lowe DM, Wilkinson AJ, Blow DM, Brick P, Carter P, Waye MMY, Winter G (1985) Nature 314:235–238PubMedCrossRefGoogle Scholar
  21. 21.
    Fersht AR (1987) Trends Bioehem Sci 12:301–304CrossRefGoogle Scholar
  22. 22.
    Fersht AR (1987) Biochemistry 26:8031–8037PubMedCrossRefGoogle Scholar
  23. 23.
    Weiner SJ, Kollman PA, Case DA, Singh UC, Ghio C, Alagona G, Profeta S, Weiner P (1984) J Amer Chem Soc 108:765–784CrossRefGoogle Scholar
  24. 24.
    Street IP, Armstrong CR, Withers SG (1986) Biochemistry 25:6021–6027PubMedCrossRefGoogle Scholar
  25. 25.
    Jones DH, McMillan AJ, Fersht AR, Winter G (1985) Biochemistry 24:5852–5857PubMedCrossRefGoogle Scholar
  26. 26.
    Ward WHJ, Jones DH, Fersht AR (1986) J Biol Chem 261:9576–9578PubMedGoogle Scholar
  27. 27.
    Ward WHJ, Jones DH, Fersht AR, (1987) Biochemistry 26:4131–4138PubMedCrossRefGoogle Scholar
  28. 28.
    Wells TNC, Fersht AR (1986) Biochemistry 25:1881–1886PubMedCrossRefGoogle Scholar
  29. 29.
    Ho C, Fersht AR (1986) Biochemistry 25:1891–1897PubMedCrossRefGoogle Scholar
  30. 30.
    Lowe DM, Winter G, Fersht AR (1987) Biochemistry 26:6038–6043PubMedCrossRefGoogle Scholar
  31. 31.
    Haidane JBS (1930) Enzymes, Longmans Green & Co Ltd, UK (Reprinted 1965 MIT Press, Cambridge, USA)Google Scholar
  32. 32.
    Pauling L (1946) Chem Eng News 24:1375–1377CrossRefGoogle Scholar
  33. 33.
    Fersht AR (1985) Enzyme structure and Mechanism, Freeman, New YorkGoogle Scholar
  34. 34.
    Leatherbarrow RJ, Fersht AR, Winter G (1985) Proc Natl Acad Sci, USA 82:7840–7844PubMedCrossRefGoogle Scholar
  35. 35.
    Bedouelle H, Winter G (1985) Nature 320:371–373CrossRefGoogle Scholar
  36. 36.
    Fersht AR, Knill-Jones J, Bedouelle H, Winter G (1987b) Biochemistry (in press)Google Scholar
  37. 37.
    Monteilhet C, Blow DM (1978) J Molec Biol 122:407–417PubMedCrossRefGoogle Scholar
  38. 38.
    Fersht AR (1975) Biochemistry 14:5–12PubMedCrossRefGoogle Scholar
  39. 39.
    Ward WHJ, Fersht AR (1988) Biochemistry (in press)Google Scholar
  40. 40.
    Schimmel P, Soll D (1979) Ann Rev Biochem 48:601–648PubMedCrossRefGoogle Scholar
  41. 41.
    Carter P, Bedouelle H, Winter G (1985) Proc Nad Acad Sci, USA 83:1189–1192CrossRefGoogle Scholar
  42. 42.
    Ward WHJ, Fersht AR (1988) (submitted for publication)Google Scholar
  43. 43.
    Albery WJ, Knowles JR (1976) Angew Chem Int Ed Engl 16: 285–293CrossRefGoogle Scholar
  44. 44.
    Fersht AR (1974) Proc R Soc London B187:397–407CrossRefGoogle Scholar
  45. 45.
    Fersht AR, Wilkinson AJ, Carter P, Winter G (1985) Biochemistry 24:5858–5861PubMedCrossRefGoogle Scholar
  46. 46.
    Thomas PG, Russell AJ, Fersht AR (1985) Nature 318:375–376CrossRefGoogle Scholar
  47. 47.
    Russell AJ, Thomas PG, Fersht, AR (1987) J Mol Biol 193:803–813PubMedCrossRefGoogle Scholar
  48. 48.
    Russe U AJ, Fersht AR (1987) Nature 328:496–500CrossRefGoogle Scholar
  49. 49.
    Sternberg MJE, Hayes FR, Russell AJ, Thomas PG, Fersht AR (1987) Nature 330:86–88PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 1988

Authors and Affiliations

  • W. H. J. Ward
    • 1
  • A. R. Fersht
    • 1
  1. 1.Department of ChemistryImperial College of Science and TechnologyLondonUK

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