iBet uBet web content aggregator. Adding the entire web to your favor.
iBet uBet web content aggregator. Adding the entire web to your favor.



Link to original content: https://doi.org/10.1038/nchem.2905
Substrate-driven chemotactic assembly in an enzyme cascade | Nature Chemistry
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.

  • Article
  • Published:

Substrate-driven chemotactic assembly in an enzyme cascade

Nature Chemistry volume 10, pages 311–317 (2018)Cite this article

Subjects

Abstract

Enzymatic catalysis is essential to cell survival. In many instances, enzymes that participate in reaction cascades have been shown to assemble into metabolons in response to the presence of the substrate for the first enzyme. However, what triggers metabolon formation has remained an open question. Through a combination of theory and experiments, we show that enzymes in a cascade can assemble via chemotaxis. We apply microfluidic and fluorescent spectroscopy techniques to study the coordinated movement of the first four enzymes of the glycolysis cascade: hexokinase, phosphoglucose isomerase, phosphofructokinase and aldolase. We show that each enzyme independently follows its own specific substrate gradient, which in turn is produced by the preceding enzymatic reaction. Furthermore, we find that the chemotactic assembly of enzymes occurs even under cytosolic crowding conditions.

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

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

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

Figure 1: Studied enzymatic reactions and microfluidic device.
Figure 2: Catalysis-induced enzyme focusing.
Figure 3: Catalysis-induced enzyme focusing and computed profiles of total enzyme concentration replicating experimental conditions from Fig. 2.
Figure 4: Chemotactic shifts observed for HK in response to gradients of different substrates.
Figure 5: Chemotactic assembly of enzymes in the microfluidic channel under different reaction conditions.
Figure 6: Examples of HK and Ald trajectories from an experiment in which D-glucose and all four enzymes were present, for which the corresponding Ald trajectory was highly correlated.

Similar content being viewed by others

References

  1. Miles, E. W., Rhee, S. & Davies, D. R. The molecular basis of substrate channeling. J. Biol. Chem. 274, 12193–12196 (1999).

    Article  CAS  Google Scholar 

  2. Wheeldon, I. et al. Substrate channelling as an approach to cascade reactions. Nat. Chem. 8, 299–309 (2016).

    Article  CAS  Google Scholar 

  3. Lin, J.-L., Palomec, L. & Wheeldon, I. Design and analysis of enhanced catalysis in scaffolded multienzyme cascade reactions. ACS Catal. 4, 505–511 (2014).

    Article  CAS  Google Scholar 

  4. Yamada, Y. et al. Nanocrystal bilayer for tandem catalysis. Nat. Chem. 3, 372–376 (2011).

    Article  CAS  Google Scholar 

  5. Zhao, M. et al. Core–shell palladium nanoparticle@metal–organic frameworks as multifunctional catalysts for cascade reactions. J. Am. Chem. Soc. 136, 1738–1741 (2014).

    Article  CAS  Google Scholar 

  6. Zhang, Y. H. Substrate channeling and enzyme complexes for biotechnological applications. Biotechnol. Adv. 29, 715–725 (2011).

    Article  CAS  Google Scholar 

  7. An, S., Kumar, R., Sheets, E. D. & Benkovic, S. J. Reversible compartmentalization of de novo purine biosynthetic complexes in living cells. Science 320, 103–106 (2008).

    Article  CAS  Google Scholar 

  8. Nishi, H., Hashimoto, K. & Panchenko, A. R. Phosphorylation in protein–protein binding: effect on stability and function. Structure 19, 1807–1815 (2011).

    Article  CAS  Google Scholar 

  9. Lindbladh, C. et al. Preparation and kinetic characterization of a fusion protein of yeast mitochondrial citrate synthase and malate-dehydrogenase. Biochemistry 33, 11692–11698 (1994).

    Article  CAS  Google Scholar 

  10. Wu, F. & Minteer, S. Krebs cycle metabolon: structural evidence of substrate channeling revealed by cross-linking and mass spectrometry. Angew. Chem. Int. Ed. 54, 1851–1854 (2015).

    Article  CAS  Google Scholar 

  11. Winkel-Shirley, B. Evidence for enzyme complexes in the phenylpropanoid and flavonoid pathways. Physiol. Plant. 107, 142–149 (1999).

    Article  CAS  Google Scholar 

  12. Jorgensen, K. et al. Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr. Opin. Plant. Biol. 8, 280–291 (2005).

    Article  CAS  Google Scholar 

  13. Graham, J. W. A. et al. Glycolytic enzymes associate dynamically with mitochondria in response to respiratory demand and support substrate channeling. Plant Cell 19, 3723–3738 (2007).

    Article  CAS  Google Scholar 

  14. Campanella, M. E., Chu, H. & Low, P. S. Assembly and regulation of a glycolytic enzyme complex on the human erythrocyte membrane. Proc. Natl Acad. Sci. USA 102, 2402–2407 (2005).

    Article  CAS  Google Scholar 

  15. Sengupta, S. et al. Enzyme molecules as nanomotors. J. Am. Chem. Soc. 135, 1406–1414 (2013).

    Article  CAS  Google Scholar 

  16. Sengupta, S. et al. DNA polymerase as a molecular motor and pump. ACS Nano 8, 2410–2418 (2014).

    Article  CAS  Google Scholar 

  17. Yu, H., Jo, K., Kounovsky, K. L., Pablo, J. J. & Schwartz, D. C. Molecular propulsion: chemical sensing and chemotaxis of DNA driven by RNA polymerase. J. Am. Chem. Soc. 131, 5722–5723 (2009).

    Article  CAS  Google Scholar 

  18. French, J. B. et al. Spatial colocalization and functional link of purinosomes with mitochondria. Science 351, 733–737 (2016).

    Article  CAS  Google Scholar 

  19. Kohnhorst, C. L. et al. Identification of a multienzyme complex for glucose metabolism in living cells. J. Biol. Chem. 292, 9191–9203 (2017).

    Article  CAS  Google Scholar 

  20. Muddana, H. S., Sengupta, S., Mallouk, T. E., Sen, A. & Butler, P. J. Substrate catalysis enhance single-enzyme diffusion. J. Am. Chem. Soc. 132, 2110–2111 (2010).

    Article  CAS  Google Scholar 

  21. Riedel, C. et al. The heat released during catalytic turnover enhances the diffusion of an enzyme. Nature 517, 227–230 (2015).

    Article  CAS  Google Scholar 

  22. Illien, P. et al. Exothermicity is not a necessary condition for enhanced diffusion of enzymes. Nano Lett. 17, 4415–4420 (2017).

    Article  CAS  Google Scholar 

  23. Myers, T. C., Nakamura, K. & Flesher, J. W. Phosphonic acid analogs of nucleoside phosphate. I. The synthesis of 5′-adenylyl methylenediphosphate, a phosphonic acid analog of ATP. J. Am. Chem. Soc. 85, 3292–3295 (1963).

    Article  CAS  Google Scholar 

  24. Paduano, L., Sartorio, R., Errico, G. D. & Vitagliano, V. Mutual diffusion in aqueous solution of ethylene glycol oligomers at 25 °C. J. Chem. Soc. Faraday Trans. 94, 2571–2576 (1998).

    Article  CAS  Google Scholar 

  25. Annunziata, O. et al. Quaternary diffusion coefficients in a protein–polymer–salt–water system determined by Rayleigh interferometry. J. Phys. Chem. B 113, 13446–13453 (2009).

    Article  CAS  Google Scholar 

  26. Vergara, A., Paduano, L. & Sartorio, R. Mechanism of protein–poly(ethylene glycol) interaction from a diffusive point of view. Macromolecules 35, 1389–1398 (2002).

    Article  CAS  Google Scholar 

  27. Vanag, V. K. & Epstein, I. R. Cross-diffusion and pattern formation in reaction–diffusion systems. Phys. Chem. Chem. Phys. 11, 897–912 (2009).

    Article  CAS  Google Scholar 

  28. Schurr, J. M., Fujimoto, B. S., Huynh, L. & Chiu, D. T. A theory of macromolecular chemotaxis. J. Phys. Chem. B 117, 7626–7652 (2013).

    Article  CAS  Google Scholar 

  29. Wilkinson, K. D. & Rose, I. A. Isotope trapping studies of yeast hexokinase during steady state catalysis. A combined rapid quench and isotope trapping technique. J. Biol. Chem. 254, 12567–12572 (1979).

    CAS  PubMed  Google Scholar 

  30. Bermingham, A., Bottomley, J. R., Derrick, W. U. & Derrick, J. P. Equilibrium and kinetic studies of substrate binding to 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase from Escherichia coli. J. Biol. Chem. 275, 17962–17967 (2000).

    Article  CAS  Google Scholar 

  31. Wu, F., Pelster, L. N. & Minteer, S. D. Krebs cycle metabolon formation: metabolite concentration gradient enhanced compartmentation of sequential enzymes. Chem. Commun. 51, 1244–1247 (2015).

    Article  CAS  Google Scholar 

  32. Vopel, T. & Makhatadze, G. I. Enzyme activity in the crowded milieu. PLoS ONE 7, e39418 (2012).

    Article  CAS  Google Scholar 

  33. Baum, M., Erdel, F., Wachsmuth, M. & Rippe, K. Retrieving the intracellular topology from multi-scale protein mobility mapping in living cells. Nat. Commun. 5, 4494 (2014).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The work was supported by Penn State MRSEC, funded by the National Science Foundation (NSF, DMR-1420620). P.J.B. acknowledges support from NSF CMMI 1334847. H.P. and H.H. acknowledge support by the Defense Threat Reduction Agency (award no. HDTRA1-14-1-0051).

Author information

Author notes

  1. Xi Zhao and Henri Palacci: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry, The Pennsylvania State University, University Park, 16802, Pennsylvania, USA

    Xi Zhao, Vinita Yadav, Michelle M. Spiering, Stephen J. Benkovic & Ayusman Sen

  2. Department of Biomedical Engineering, Columbia University, New York, 10027, New York, USA

    Henri Palacci & Henry Hess

  3. Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, 92093, California, USA

    Michael K. Gilson

  4. Department of Biomedical Engineering, The Pennsylvania State University, University Park, 16802, Pennsylvania, USA

    Peter J. Butler

Authors
  1. Xi Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Henri Palacci
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Vinita Yadav
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Michelle M. Spiering
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Michael K. Gilson
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Peter J. Butler
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Henry Hess
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Stephen J. Benkovic
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Ayusman Sen
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.K.G., P.J.B., H.H., S.J.B. and A.S. designed the research. X.Z., V.Y. and M.M.S. performed the experiments. H.P. performed the modelling. X.Z., H.P., V.Y., M.M.S., H.H., S.J.B. and A.S. analysed the data and wrote the manuscript. All authors contributed to the discussion of results.

Corresponding authors

Correspondence to Henry Hess, Stephen J. Benkovic or Ayusman Sen.

Ethics declarations

Competing interests

M.K.G. has an equity interest in and is a cofounder and scientific advisor of VeraChem LLC.

Supplementary information

Supplementary information

Supplementary information (PDF 1753 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhao, X., Palacci, H., Yadav, V. et al. Substrate-driven chemotactic assembly in an enzyme cascade. Nature Chem 10, 311–317 (2018). https://doi.org/10.1038/nchem.2905

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchem.2905

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