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Probing the transition state in enzyme catalysis by high-pressure NMR dynamics

Nature Catalysis volume 2, pages 726–734 (2019)Cite this article

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Abstract

Protein conformational changes are often essential for enzyme catalysis and, in several cases, are shown to be the limiting factor for overall catalytic speed. However, a structural understanding of the corresponding transition states, needed to rationalize the kinetics, remains obscure due to their transient nature. Here, we determine the transition state ensemble of the rate-limiting conformational transition in the enzyme adenylate kinase through a synergistic approach between experimental high-pressure NMR relaxation during catalysis and molecular dynamics simulations. By comparing homologous kinases that evolved under ambient or high pressure in the deep sea, we detail transition state ensembles that differ in solvation as directly measured by the pressure dependence of catalysis. Capturing transition state ensembles begins to complete the catalytic energy landscape that is generally characterized by the structures of all intermediates and the frequencies of transitions among them.

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Fig. 1: Investigating the pressure dependence of turnover for mesoAdk versus piezoAdk to gain insight into transition states.
Fig. 2: The dynamic and structural differences of Adk under pressure.
Fig. 3: The putative TSE identified from differential solvation in TMD simulations.
Fig. 4: Triple mutation induces full pressure activation in mesoAdk.

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Data availability

Structure factors and refined model of piezoAdk has been deposited in the PDB under the accession code 4K46. The data that support the plots within this paper and other findings of this study are available from the corresponding author on reasonable request.

Code availability

In-house Python scripts are available from the corresponding author on request.

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Acknowledgements

We are grateful for D. V. Pachov’s preliminary TMD simulations on mesoAdk and piezoAdk and discussions, F. Pontiggia with guidance for the TMD simulations and J. Wand and R. Peterson for the initial exploratory pressure NMR experiments. Finally, we thank D. Bartlett for generously gifting P. profundom genomic DNA. This work was supported by the Howard Hughes Medical Institute, the Office of Basic Energy Sciences, the Catalysis Science Program, the US Department of Energy (grant no. DE-FG02-05ER15699 to D.K. and NIH; grant no. GM100966 to M.F.H and D.K). R.O. was supported as an HHMI fellow of the Damon Runyon Cancer Research Foundation (grant no. DRG-2114-12). Computational resources were provided by NSF XSEDE computing resources (grant no. TG-MCB090163) and the Brandeis HPCC, which is partially supported by grant no. DMR-1420382. We thank the Advanced Light Source (ALS) for beamline use. The Berkeley Center for Structural Biology is supported in part by the National Institutes of Health, National Institute of General Medical Sciences and the HHMI. The ALS is supported by the director (Office of Science, Office of Basic Energy Sciences) of the US Department of Energy under contract no. DE-AC02-05CH11231.

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Author notes

  1. S. Jordan Kerns

    Present address: Emulate Inc., Boston, MA, USA

  2. These authors contributed equally: John B. Stiller, S. Jordan Kerns.

Authors and Affiliations

  1. Department of Biochemistry and Howard Hughes Medical Institute, Brandeis University, Waltham, MA, USA

    John B. Stiller, S. Jordan Kerns, Marc Hoemberger, Young-Jin Cho, Renee Otten & Dorothee Kern

  2. Department of Physics, Brandeis University, Waltham, MA, USA

    Michael F. Hagan

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Contributions

D.K. conceived the project idea. D.K., S.J.K, and M.F.H. developed the research plan and experimental strategy. S.J.K performed pressure-variable kinetic experiments, which included purifying the wild-type and mutant proteins, designing and assembling the high-pressure spectrophotometer, performing pressure-variable experiments and analysing the results. S.J.K, with help from R.O., also performed all NMR experiments. J.B.S, S.J.K. and R.O. performed the NMR data analyses, fitting and interpretation. Y.J.-C. purified piezoAdk for crystallization and solved the X-ray structure. M.H. performed the TMD simulations. M.H. and J.B.S analysed the TMD simulations with assistance by M.F.H. and D.K. S.J.K. and D.K. designed mutations on the basis of experimental data and TMD simulations. All authors discussed the results, which led to the overall scientific findings. J.B.S. and D.K wrote the manuscript. All authors reviewed and edited the manuscript.

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Correspondence to Dorothee Kern.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–10, Supplementary Tables 1 and 2, Supplementary Note 1 and Supplementary References

Reporting Summary

Supplementary Data 1

Triple mutant mesoAdk final structure

Supplementary Data 2

MesoAdk final structure

Supplementary Data 3

MesoAdk initial structure

Supplementary Data 4

PiezoAdk final structure

Supplementary Data 5

PiezoAdk initial structure

Supplementary Data 6

Triple mutant mesoAdk initial structure

Supplementary Video 1

Hydration of AMP-lid interface in piezoAdk

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Stiller, J.B., Jordan Kerns, S., Hoemberger, M. et al. Probing the transition state in enzyme catalysis by high-pressure NMR dynamics. Nat Catal 2, 726–734 (2019). https://doi.org/10.1038/s41929-019-0307-6

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