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Substrate-driven chemotactic assembly in an enzyme cascade

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.

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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.

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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).

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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.

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Competing interests

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

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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

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