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The heat released during catalytic turnover enhances the diffusion of an enzyme

Nature volume 517, pages 227–230 (2015)Cite this article

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Abstract

Recent studies have shown that the diffusivity of enzymes increases in a substrate-dependent manner during catalysis1,2. Although this observation has been reported and characterized for several different systems3,4,5,6,7,8,9,10, the precise origin of this phenomenon is unknown. Calorimetric methods are often used to determine enthalpies from enzyme-catalysed reactions and can therefore provide important insight into their reaction mechanisms11,12. The ensemble averages involved in traditional bulk calorimetry cannot probe the transient effects that the energy exchanged in a reaction may have on the catalyst. Here we obtain single-molecule fluorescence correlation spectroscopy data and analyse them within the framework of a stochastic theory to demonstrate a mechanistic link between the enhanced diffusion of a single enzyme molecule and the heat released in the reaction. We propose that the heat released during catalysis generates an asymmetric pressure wave that results in a differential stress at the protein–solvent interface that transiently displaces the centre-of-mass of the enzyme (chemoacoustic effect). This novel perspective on how enzymes respond to the energy released during catalysis suggests a possible effect of the heat of reaction on the structural integrity and internal degrees of freedom of the enzyme.

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Figure 1: FCS data and residuals of the fits.
Figure 2: Enhanced diffusion as a function of the reaction rate.

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Change history

  • 07 January 2015

    A minor change was made to Fig. 1 and an additional affiliation included for author C.B.

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Acknowledgements

We thank A. Sen for discussions of his experiments. We thank J. Kirsch, R. Golestanian, D. Leitner, J. Kirsdu, P. Geissler, P. Nelson and A. Szabo for their feedback on this work, J. Liphardt for the 405-nm laser line, P. Harbury for the TIM plasmid and B. Maguire for the TIM expression. This research was supported in part by NIH grants R01-GM0325543 (C.B.) and R01-GM05945 (S.M.), the US Department of Energy, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering under contract no. DE-AC02-05CH11231 (C.B.), and the NSF grants MCB-1412259 (S.P.) and MCB-1122225 (S.M.). K.M.H. was supported in part by grant NIGMS, R01-GM65050. C.R. acknowledges the support of the Human Frontier Science Program. S.P. further acknowledges support from the Burroughs-Wellcome Fund.

Author information

Author notes

  1. Kambiz Hamadani

    Present address: Present address: Department of Chemistry and Biochemistry, California State University San Marcos, California 92078, USA.,

Authors and Affiliations

  1. California Institute for Quantitative Biosciences, QB3, University of California, Berkeley, 94720, California, USA

    Clement Riedel, Ronen Gabizon, Christian A. M. Wilson, Kambiz Hamadani, Susan Marqusee & Carlos Bustamante

  2. Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, 1058 Santiago, Chile,

    Christian A. M. Wilson

  3. Department of Physics, Indiana University-Purdue University Indianapolis (IUPUI), 46202, Indiana, USA

    Konstantinos Tsekouras & Steve Pressé

  4. Department of Molecular and Cell Biology, University of California, Berkeley, 94720, California, USA

    Susan Marqusee & Carlos Bustamante

  5. Department of Cellular and Integrative Physiology, Indiana University School of Medicine, 46202, Indiana, USA

    Steve Pressé

  6. Jason L. Choy Laboratory of Single-Molecule Biophysics and Department of Physics, University of California, Berkeley, 94720, California, USA

    Carlos Bustamante

  7. Department of Chemistry, University of California, Berkeley, 94720, California, USA

    Carlos Bustamante

  8. Howard Hughes Medical Institute, University of California, Berkeley, 94720, California, USA

    Carlos Bustamante

  9. Kavli Energy Nano Sciences Institute, University of California, Berkeley and Lawrence Berkeley National Laboratory, 94720, California, USA

    Carlos Bustamante

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Contributions

C.R. performed fluorescence correlation spectroscopy (FCS) measurements, assisted in most experiments and was the primary writer of the manuscript. R.G. and C.A.M.W. ran most bulk biochemistry experiments; K.H. built the FCS setup and assisted in the FCS experiments; S.M. gave direction to the project. S.P. supervised the research and S.P. and K.T. developed the theory. Finally C.B. conceived the project, and supervised all of the research. All authors participated in the writing and editing of the manuscript.

Corresponding authors

Correspondence to Steve Pressé or Carlos Bustamante.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Reaction rate per enzyme molecule as a function of the concentration of substrate.

a, Catalase; b, urease; c, alkaline phosphatase and d, triose phosphate isomerase. Lines are fit to a Michaelis–Menten curve. Error bars represent the standard deviation of 3 measurements.

Extended Data Figure 2 Isothermal titration calorimetry measurement of the heat of hydrolysis of p-nitrophenylphosphate by alkaline phosphatase.

Each peak corresponds to the hydrolysis of 0.053 µmol of substrate. The reaction rate slows as more substrate is added owing to the accumulation of phosphate product inhibiting the enzyme.

Extended Data Figure 3 Diffusion coefficient of non-reactive urease in the presence of active, non-labelled, catalase (1 nM) for different concentrations of hydrogen peroxide.

Even at the highest catalase activity no indirect effects (due to bubbling or global heating of the solution) appreciably increase the diffusion coefficient of freely diffusing urease. Error bars are computed from the standard deviation over 10 measurements.

Extended Data Figure 4 Haem excitation experiment.

a, Diffusion coefficient of catalase as a function of the laser power at 402 nm. The heat released by the haem enhances the diffusion of the enzyme. b, Diffusion coefficient of free dyes in the presence of non-labelled heat-emitting catalase. Neither the heat generated by the laser power nor the heat released by catalase significantly enhances the diffusion of the dyes. Error bars are computed from the standard deviation over 10 measurements.

Extended Data Figure 5 Structures of the enzymes studied with active site components highlighted.

Different shades of grey indicate different monomers. a, Catalase (PDB 3NWL). Haem groups are shown as red sticks. b, Urease (PDB 4GY7). Active site nickel ions are shown as green spheres. c, Alkaline phosphatase (PDB 4KJG). Magnesium ions are shown in green; zinc ions are shown in magenta; 4-nitrophenol (hydrolysis product bound at the active site) is shown as yellow sticks. d, Triose phosphate isomerase (8TIM). Active site residues Lys 12, His 95 and Glu 165 are shown as yellow sticks.

Extended Data Figure 6 Protein deformation progression.

Spread of a radial deformation wave (red) emanating from the catalytic site (yellow star). The protein (grey) has an approximate radius of 4 nm and the deformation wave speed is an estimated 3 nm ps−1.

Extended Data Figure 7 Centre of mass translation.

The protein exerts forces on the solvent (small blue arrows). The solvent restoring forces (thick blue arrows) give rise to a protein centre of mass translation (black arrow).

Extended Data Figure 8 Acoustic wave due to deformation.

Here we assume the protein deforms and this generates acoustic waves in the solvent. If these acoustic waves were reflected back on the protein, they would give rise to a centre of mass translation denoted by the blue arrow.

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Riedel, C., Gabizon, R., Wilson, C. et al. The heat released during catalytic turnover enhances the diffusion of an enzyme. Nature 517, 227–230 (2015). https://doi.org/10.1038/nature14043

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