Single Enzyme Studies: A Historical Perspective

  • Alex E. KnightEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 778)


Single-molecule enzymology has a longer history than is often supposed, with the first measurements being made as early as 1961. However, the development of new technologies has meant that most of the progress has been made in the last two decades. I review the development of single-molecule enzymology, focussing on five key papers which are milestones in the field. In particular, I discuss the 1961 paper by Boris Rotman, which made inventive use of what now seems like primitive technology, and continues to be influential to this day.

Key words

Single-molecule enzymology Static heterogeneity Dynamic heterogeneity Single turnovers 



I would like to acknowledge support from the Chemical and Biological Metrology programme of the National Measurement Office.


  1. 1.
    Rotman, B. (1961) Measurement of activity of single molecules of β-D-galactosidase. Proc. Natl. Acad. Sci. USA 47, 1981–1991.PubMedCrossRefGoogle Scholar
  2. 2.
    Weiss, S. (1999) Fluorescence Spectroscopy of Single Biomolecules, Science 283, 1676–1683.PubMedCrossRefGoogle Scholar
  3. 3.
    Knight, A. E., (Ed.) (2009) Single Molecule Biology Academic Press.Google Scholar
  4. 4.
    Bustamante, C. (2008) In singulo Biochemistry: When Less Is More. Annu. Rev. Biochem. 77, 45–50.PubMedCrossRefGoogle Scholar
  5. 5.
    Moerner, W. E. (2007) New directions in single-molecule imaging and analysis. Proc. Natl. Acad. Sci. USA 104, 12596–12602.PubMedCrossRefGoogle Scholar
  6. 6.
    Millikan, R. A. (1913) On the Elementary Electrical Charge and the Avogadro Constant. Phys. Rev. 2, 109.CrossRefGoogle Scholar
  7. 7.
    Cai, L., Friedman, N., and Xie, X. S. (2006) Stochastic protein expression in individual cells at the single molecule level. Nature 440, 358–362.PubMedCrossRefGoogle Scholar
  8. 8.
    Xue, Q., and Yeung, E. S. (1995) Differences in the chemical reactivity of individual molecules of an enzyme. Nature 373, 681–683.PubMedCrossRefGoogle Scholar
  9. 9.
    Funatsu, T., Harada, Y., Tokunaga, M., Saito, K., and Yanagida, T. (1995) Imaging of Single Fluorescent Molecules and Individual ATP Turnovers by Single Myosin Molecules in Aqueous Solution. Nature 374, 555–559.PubMedCrossRefGoogle Scholar
  10. 10.
    Svoboda, K., Schmidt, C. F., Schnapp, B. J., and Block, S. M. (1993) Direct observation of kinesin stepping by optical trapping interferometry. Nature 365, 721–727.PubMedCrossRefGoogle Scholar
  11. 11.
    Finer, J. T., Simmons, R. M., and Spudich, J. A. (1994) Single Myosin Molecule Mechanics – Piconewton Forces and Nanometer Steps. Nature 368, 113–119.PubMedCrossRefGoogle Scholar
  12. 12.
    Knight, A., Mashanov, G., and Molloy, J. (2005) Single molecule measurements and biological motors. Eur. Biophys. J. 35, 89.PubMedCrossRefGoogle Scholar
  13. 13.
    Lu, H. P., Xun, L., and Xie, X. S. (1998) Single-Molecule Enzymatic Dynamics. Science 282, 1877–1882.PubMedCrossRefGoogle Scholar
  14. 14.
    English, B. P., Min, W., van Oijen, A. M., Lee, K. T., Luo, G., Sun, H., Cherayil, B. J., Kou, S. C., and Xie, X. S. (2006) Ever-fluctuating single enzyme molecules: Michaelis-Menten equation revisited. Nat. Chem. Biol. 2, 87.PubMedCrossRefGoogle Scholar
  15. 15.
    Visscher, K., Schnitzer, M., and Block, S. (1999) Single kinesin molecules studied with a molecular force clamp. Nature 400, 184–189.PubMedCrossRefGoogle Scholar
  16. 16.
    Puchner, E. M., Alexandrovich, A., Kho, A. L., Hensen, U., Schäfer, L., Brandmeier, B., Gräter, F., Grubmüller, H., Gaub, H., and Gautel, M. (2008) Mechanoenzymatics of titin kinase. Proc. Natl. Acad. Sci. USA 105, 13385–13390.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Analytical Science DivisionNational Physical LaboratoryTeddingtonUK

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