Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Perspective
  • Published:

A 21st century revisionist's view at a turning point in enzymology

Nature Chemical Biology volume 5, pages 543–550 (2009)Cite this article

A Corrigendum to this article was published on 01 September 2009

This article has been updated

Abstract

Despite the fact that the number of publications associated with the keyword 'enzyme' increases every year, the precise origin of enzyme catalysis has remained unresolved. Because of sustained intensive research efforts from an increasing number of laboratories, detailed information regarding the physics, chemistry and kinetics of enzymes is accumulating rapidly. The growing body of data contains many examples of kinetic behavior that are incompatible with a static view of enzyme catalysis. As a result, numerous laboratories are approaching the consensus that protein motion plays an essential role in enzyme catalysis. A model that incorporates nuclear quantum tunneling together with two classes of protein motion—termed conformational sampling (pre-organization) and reorganization—is recommended as a means of understanding the large body of data for enzyme-catalyzed hydrogen transfers. It should also serve as a vehicle for future efforts in the development of potent enzyme inhibitors and the de novo design of all classifications of enzymes.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: An Arrhenius plot shows the temperature dependence of a simulated hydrogen transfer reaction.
Figure 2: A basic representation of the tunneling phenomenon is illustrated.
Figure 3: A full tunneling model for hydrogen transfer is represented.
Figure 4: Impact of pre-organization on reorganization.

Similar content being viewed by others

Change history

  • 20 July 2009

    In the version of this article initially published, in the first line on the right hand column of Box 2, the sentence began "The distance ro...," whereas it should read "The tunneling distance...." Also, reference 47 was omitted from the end of the second to last sentence of the legend of Figure 3. These errors have been corrected in the HTML and PDF versions of the article.

References

  1. Pauling, L. Molecular architecture and biological reactions. Chem. Eng. News 24, 1375 (1946).

    Article  CAS  Google Scholar 

  2. Blake, C.C.F. et al. Structure of hen egg-white lysozyme - a 3-dimensional Fourier synthesis at 2Å resolution. Nature 206, 757–761 (1965).

    Article  CAS  PubMed  Google Scholar 

  3. Monod, J., Wyman, J. & Changeux, J.P. On the nature of allosteric transitions - a plausible model. J. Mol. Biol. 12, 88–118 (1965).

    Article  CAS  PubMed  Google Scholar 

  4. Kaptein, R., Zuiderweg, E.R.P., Scheek, R.M., Boelens, R. & Vangunsteren, W.F. A protein structure from nuclear magnetic-resonance data - Lac repressor headpiece. J. Mol. Biol. 182, 179–182 (1985).

    Article  CAS  PubMed  Google Scholar 

  5. Cha, Y., Murray, C.J. & Klinman, J.P. Hydrogen tunneling in enzyme-reactions. Science 243, 1325–1330 (1989). This was the first clear demonstration of significant room temperature tunneling in an enzyme-catalyzed reaction.

    Article  CAS  PubMed  Google Scholar 

  6. Klinman, J.P. An integrated model for enzyme catalysis emerges from studies of hydrogen tunneling. Chem. Phys. Lett. 471, 179–193 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Jencks, W.P. Catalysis in Chemistry and Enzymology (McGraw-Hill, New York, 1969).

    Google Scholar 

  8. Pollack, S.J., Jacobs, J.W. & Schultz, P.B. Selective chemical catalysis by an antibody. Science 234, 1570–1573 (1986).

    Article  CAS  PubMed  Google Scholar 

  9. Hilvert, D. Critical analysis of antibody catalysis. Annu. Rev. Biochem. 69, 751–793 (2000).

    Article  CAS  PubMed  Google Scholar 

  10. Jäckel, C., Kast, P. & Hilvert, D. Protein design by directed evolution. Annu. Rev. Biophys. 37, 153–173 (2008).

    Article  PubMed  Google Scholar 

  11. Lin, H.N., Tao, H.Y. & Cornish, V.W. Directed evolution of a glycosynthase via chemical complementation. J. Am. Chem. Soc. 126, 15051–15059 (2004).

    Article  CAS  PubMed  Google Scholar 

  12. Griffiths, A.D. & Tawfik, D.S. Man-made enzymes - from design to in vitro compartmentalisation. Curr. Opin. Biotechnol. 11, 338–353 (2000).

    Article  CAS  PubMed  Google Scholar 

  13. Taylor, S.V., Kast, P. & Hilvert, D. Investigating and engineering enzymes by genetic selection. Angew. Chem. Int. Ed. 40, 3310–3335 (2001).

    Article  Google Scholar 

  14. Röthlisberger, D. et al. Kemp elimination catalysts by computational enzyme design. Nature 453, 190–195 (2008).

    Article  PubMed  Google Scholar 

  15. Voigt, C.A., Gordon, D.B. & Mayo, S.L. Trading accuracy for speed: A quantitative comparison of search algorithms in protein sequence design. J. Mol. Biol. 299, 789–803 (2000).

    Article  CAS  PubMed  Google Scholar 

  16. McCammon, J. & Harvey, S. Dynamics of Proteins and Nucleic Acids, 234 (Press Syndicate of the University of Cambridge, New York, 1987).

    Book  Google Scholar 

  17. English, B.P. et al. Ever-fluctuating single enzyme molecules: Michaelis-Menten equation revisited. Nat. Chem. Biol. 2, 87–94 (2006). Single-molecule Michaelis Menten kinetics demonstrated a large number of rate constants contributing to observed rate constant in bulk measurements.

    Article  CAS  PubMed  Google Scholar 

  18. Lu, H.P., Xun, L.Y. & Xie, X.S. Single-molecule enzymatic dynamics. Science 282, 1877–1882 (1998).

    Article  CAS  PubMed  Google Scholar 

  19. Pudney, C.R. et al. Mutagenesis of morphinone reductase induces multiple reactive configurations and identifies potential ambiguity in kinetic analysis of enzyme tunneling mechanisms. J. Am. Chem. Soc. 129, 13949–13956 (2007).

    Article  CAS  PubMed  Google Scholar 

  20. Liang, Z.X., Lee, T., Resing, K.A., Ahn, N.G. & Klinman, J.P. Thermal-activated protein mobility and its correlation with catalysis in thermophilic alcohol dehydrogenase. Proc. Natl. Acad. Sci. USA 101, 9556–9561 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Kohen, A., Cannio, R., Bartolucci, S. & Klinman, J.P. Enzyme dynamics and hydrogen tunnelling in a thermophilic alcohol dehydrogenase. Nature 399, 496–499 (1999). A thermophilic enzyme showed most efficient tunneling at elevated temperature. This stood in stark contrast to predictions that tunneling should become less important as temperature increases.

    Article  CAS  PubMed  Google Scholar 

  22. Maglia, G. & Allemann, R.K. Evidence for environmentally coupled hydrogen tunneling during dihydrofolate reductase catalysis. J. Am. Chem. Soc. 125, 13372–13373 (2003).

    Article  CAS  PubMed  Google Scholar 

  23. Heyes, D.J., Sakuma, M., de Visser, S.P. & Scrutton, N.S. Nuclear quantum tunneling in the light-activated enzyme protochlorophyllide oxidoreductase. J. Biol. Chem. 284, 3762–3767 (2009).

    Article  CAS  PubMed  Google Scholar 

  24. Eyring, H. The activated complex in chemical reactions. J. Chem. Phys. 3, 107–115 (1935).

    Article  CAS  Google Scholar 

  25. Bell, R.P. The Tunneling Effect in Chemistry (Chapman and Hall, New York, 1980).

    Book  Google Scholar 

  26. Garrett, B.C. & Truhlar, D.G. Criterion of minimum state density in the transition-state theory of bimolecular reactions. J. Chem. Phys. 70, 1593–1598 (1979).

    Article  CAS  Google Scholar 

  27. Truhlar, D.G. et al. Ensemble-averaged variational transition state theory with optimized multidimensional tunneling for enzyme kinetics and other condensed-phase reactions. Int. J. Quantum Chem. 100, 1136–1152 (2004).

    Article  CAS  Google Scholar 

  28. Glickman, M.H. & Klinman, J.P. Nature of rate-limiting steps in the soybean lipoxygenase-1 reaction. Biochemistry 34, 14077–14092 (1995).

    Article  CAS  PubMed  Google Scholar 

  29. Nesheim, J.C. & Lipscomb, J.D. Large kinetic isotope effects in methane oxidation catalyzed by methane monooxygenase: evidence for C-H bond cleavage in a reaction cycle intermediate. Biochemistry 35, 10240–10247 (1996).

    Article  CAS  PubMed  Google Scholar 

  30. Melander, L. & Saunders, W.H. Reaction Rates of Isotopic Molecules, 331 (Robert E. Krieger Publishing Company, Malabar, India, 1987).

    Google Scholar 

  31. Knapp, M.J., Rickert, K. & Klinman, J.P. Temperature-dependent isotope effects in soybean lipoxygenase-1: correlating hydrogen tunneling with protein dynamics. J. Am. Chem. Soc. 124, 3865–3874 (2002). A full tunneling model was derived from earlier work by Kuznetsov and Ulstrup (ref. 32 ) and used to reproduce temperature-dependent KIEs in soybean lipoxygenase.

    Article  CAS  PubMed  Google Scholar 

  32. Kuznetsov, A.M. & Ulstrup, J. Proton and hydrogen atom tunnelling in hydrolytic and redox enzyme catalysis. Can. J. Chem. 77, 1085–1096 (1999).

    CAS  Google Scholar 

  33. Meyer, M.P. & Klinman, J.P. Modeling temperature dependent kinetic isotope effects for hydrogen transfer in a series of soybean lipoxygenase mutants: the effect of anharmonicity upon transfer distance. Chem. Phys. 319, 283–296 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Hothi, P. et al. Driving force analysis of proton tunnelling across a reactivity series for an enzyme-substrate complex. ChemBioChem 9, 2839–2845 (2008).

    Article  CAS  PubMed  Google Scholar 

  35. Schramm, V.L. Enzymatic transition states: thermodynamics, dynamics and analogue design. Arch. Biochem. Biophys. 433, 13–26 (2005).

    Article  CAS  PubMed  Google Scholar 

  36. Saen-Oon, S., Quaytman-Machleder, S., Schramm, V.L. & Schwartz, S.D. Atomic detail of chemical transformation at the transition state of an enzymatic reaction. Proc. Natl. Acad. Sci. USA 105, 16543–16548 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Boehr, D.D., Dyson, H.J. & Wright, P.E. Conformational relaxation following hydride transfer plays a limiting role in dihydrofolate reductase catalysis. Biochemistry 47, 9227–9233 (2008).

    Article  CAS  PubMed  Google Scholar 

  38. McElheny, D., Schnell, J.R., Lansing, J.C., Dyson, H.J. & Wright, P.E. Defining the role of active-site loop fluctuations in dihydrofolate reductase catalysis. Proc. Natl. Acad. Sci. USA 102, 5032–5037 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Henzler-Wildman, K. & Kern, D. Dynamic personalities of proteins. Nature 450, 964–972 (2007).

    Article  CAS  PubMed  Google Scholar 

  40. Henzler-Wildman, K.A. et al. A hierarchy of timescales in protein dynamics is linked to enzyme catalysis. Nature 450, 913–916 (2007).

    Article  CAS  PubMed  Google Scholar 

  41. Min, W., Xie, X.S. & Bagchi, B. Two-dimensional reaction free energy surfaces of catalytic reaction: effects of protein conformational dynamics on enzyme catalysis. J. Phys. Chem. B 112, 454–466 (2008).

    Article  CAS  PubMed  Google Scholar 

  42. Saen-Oon, S., Ghanem, M., Schramm, V.L. & Schwartz, S.D. Remote mutations and active site dynamics correlate with catalytic properties of purine nucleoside phosphorylase. Biophys. J. 94, 4078–4088 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Kohen, A., Jonsson, T. & Klinman, J.P. Effects of protein glycosylation on catalysis: changes in hydrogen tunneling and enthalpy of activation in the glucose oxidase reaction. Biochemistry 36, 2603–2611 (1997).

    Article  CAS  PubMed  Google Scholar 

  44. Seymour, S.L. & Klinman, J.P. Comparison of rates and kinetic isotope effects using PEG-modified variants and glycoforms of glucose oxidase: the relationship of modification of the protein envelope to C-H activation and tunneling. Biochemistry 41, 8747–8758 (2002).

    Article  CAS  PubMed  Google Scholar 

  45. Tsai, S.C. & Klinman, J.P. Probes of hydrogen tunneling with horse liver alcohol dehydrogenase at subzero temperatures. Biochemistry 40, 2303–2311 (2001).

    Article  CAS  PubMed  Google Scholar 

  46. Wang, L., Goodey, N.M., Benkovic, S.J. & Kohen, A. Coordinated effects of distal mutations on environmentally coupled tunneling in dihydrofolate reductase. Proc. Natl. Acad. Sci. USA 103, 15753–15758 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Meyer, M.P., Tomchick, D.R. & Klinman, J.P. Enzyme structure and dynamics affect hydrogen tunneling: the impact of a remote side chain (1553) in soybean lipoxygenase-1. Proc. Natl. Acad. Sci. USA 105, 1146–1151 (2008). A distal mutation in SLO produced temperature-dependent KIEs. X-ray structures of the mutant enzymes showed no changes, implying that dynamical changes are required to account for the kinetic behavior.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Liu, H.B. & Warshel, A. Origin of the temperature dependence of isotope effects in enzymatic reactions: the case of dihydrofolate reductase. J. Phys. Chem. B 111, 7852–7861 (2007).

    Article  CAS  PubMed  Google Scholar 

  49. Roca, M., Messer, B., Hilvert, D. & Warshel, A. On the relationship between folding and chemical landscapes in enzyme catalysis. Proc. Natl. Acad. Sci. USA 105, 13877–13882 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Radkiewicz, J.L. & Brooks, C.L. Protein dynamics in enzymatic catalysis: exploration of dihydrofolate reductase. J. Am. Chem. Soc. 122, 225–231 (2000).

    Article  CAS  Google Scholar 

  51. Truhlar, D.G. et al. Ensemble-averaged variational transition state theory with optimized multidimensional tunneling for enzyme kinetics and other condensed-phase reactions. Int. J. Quantum Chem. 100, 1136–1152 (2004).

    Article  CAS  Google Scholar 

  52. Benkovic, S.J., Hammes, G.G. & Hammes-Schiffer, S. Free-energy landscape of enzyme catalysis. Biochemistry 47, 3317–3321 (2008).

    Article  CAS  PubMed  Google Scholar 

  53. Limbach, H.H., Lopez, J.M. & Kohen, A. Arrhenius curves of hydrogen transfers: tunnel effects, isotope effects and effects of pre-equilibria. Philos. Trans. R. Soc. Lond., B, Biol. Sci. 361, 1399–1415 (2006).

    Article  CAS  Google Scholar 

  54. Zhang, Z.Q., Rajagopalan, P.T.R., Selzer, T., Benkovic, S.J. & Hammes, G.G. Single-molecule and transient kinetics investigation of the interaction of dihydrofolate reductase with NADPH and dihydrofolate. Proc. Natl. Acad. Sci. USA 101, 2764–2769 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Hammes-Schiffer, S. & Benkovic, S.J. Relating protein motion to catalysis. Annu. Rev. Biochem. 75, 519–541 (2006).

    Article  CAS  PubMed  Google Scholar 

  56. Wang, L., Goodey, N.M., Benkovic, S.J. & Kohen, A. The role of enzyme dynamics and tunnelling in catalysing hydride transfer: studies of distal mutants of dihydrofolate reductase. Philos. Trans. R. Soc. Lond., B, Biol. Sci. 361, 1307–1315 (2006). A distal mutation in DHFR led to temperature-dependent KIEs. This was surprising because of the distance between a relatively conservative M-W mutation and the active site of the enzyme.

    Article  CAS  Google Scholar 

  57. Wolf-Watz, M. et al. Linkage between dynamics and catalysis in a thermophilic-mesophilic enzyme pair. Nat. Struct. Mol. Biol. 11, 945–949 (2004).

    Article  CAS  PubMed  Google Scholar 

  58. Gerhart, J.C. & Schachman, H.K. Allosteric interactions in aspartate transcarba-mylase 2. Evidence for different conformational states of protein in presence and absence of specific ligands. Biochemistry 7, 538–552 (1968).

    Article  CAS  PubMed  Google Scholar 

  59. Schnell, J.R., Dyson, H.J. & Wright, P.E. Effect of cofactor binding and loop conformation on side chain methyl dynamics in dihydrofolate reductase. Biochemistry 43, 374–383 (2004).

    Article  CAS  PubMed  Google Scholar 

  60. Marcus, R.A. & Sutin, N. Electron transfers in chemistry and biology. Biochim. Biophys. Acta 811, 265–322 (1985).

    Article  CAS  Google Scholar 

  61. Pang, J.Y., Hay, S., Scrutton, N.S. & Sutcliffe, M.J. Deep tunneling dominates the biologically important hydride transfer reaction from NADH to FMN in morphinone reductase. J. Am. Chem. Soc. 130, 7092–7097 (2008).

    Article  CAS  PubMed  Google Scholar 

  62. Basran, J., Sutcliffe, M.J. & Scrutton, N.S. Enzymatic H-transfer requires vibration-driven extreme tunneling. Biochemistry 38, 3218–3222 (1999).

    Article  CAS  PubMed  Google Scholar 

  63. Harris, R.J., Meskys, R., Sutcliffe, M.J. & Scrutton, N.S. Kinetic studies of the mechanism of carbon-hydrogen bond breakage by the heterotetrameric sarcosine oxidase of Arthrobacter sp 1-IN. Biochemistry 39, 1189–1198 (2000).

    Article  CAS  PubMed  Google Scholar 

  64. Fan, F. & Gadda, G. An internal equilibrium preorganizes the enzyme-substrate complex for hydride tunneling in choline oxidase. Biochemistry 46, 6402–6408 (2007).

    Article  CAS  PubMed  Google Scholar 

  65. Swain, C.G., Stivers, E.C., Reuwer, J.F. & Schaad, L.J. Use of hydrogen isotope effects to identify the attacking nucleophile in the enolization of ketones catalyzed by acetic acid. J. Am. Chem. Soc. 80, 5885–5893 (1958).

    Article  CAS  Google Scholar 

  66. Haslett, J.W. Translation of phase waves of de Broglie. Am. J. Phys. 41, 445 (1973).

    Article  Google Scholar 

  67. Marcus, R.A. Theory of oxidation-reduction reactions involving electron transfer. 1. J. Chem. Phys. 24, 966–978 (1956).

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Zachary D. Nagel is in the Department of Chemistry and Judith P. Klinman is in the Department of Chemistry and Department of Molecular and Cell Biology, University of California, Berkeley, California, USA.,

    Zachary D Nagel & Judith P Klinman

Authors
  1. Zachary D Nagel
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Judith P Klinman
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Judith P Klinman.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Nagel, Z., Klinman, J. A 21st century revisionist's view at a turning point in enzymology. Nat Chem Biol 5, 543–550 (2009). https://doi.org/10.1038/nchembio.204

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.204

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing