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Probing the chemistry of thioredoxin catalysis with force

Abstract

Thioredoxins are enzymes that catalyse disulphide bond reduction in all living organisms1. Although catalysis is thought to proceed through a substitution nucleophilic bimolecular (SN2) reaction1,2, the role of the enzyme in modulating this chemical reaction is unknown. Here, using single-molecule force-clamp spectroscopy3,4, we investigate the catalytic mechanism of Escherichia coli thioredoxin (Trx). We applied mechanical force in the range of 25–600 pN to a disulphide bond substrate and monitored the reduction of these bonds by individual enzymes. We detected two alternative forms of the catalytic reaction, the first requiring a reorientation of the substrate disulphide bond, causing a shortening of the substrate polypeptide by 0.79 ± 0.09 Å (± s.e.m.), and the second elongating the substrate disulphide bond by 0.17 ± 0.02 Å (± s.e.m.). These results support the view that the Trx active site regulates the geometry of the participating sulphur atoms with sub-ångström precision to achieve efficient catalysis. Our results indicate that substrate conformational changes may be important in the regulation of Trx activity under conditions of oxidative stress and mechanical injury, such as those experienced in cardiovascular disease5,6. Furthermore, single-molecule atomic force microscopy techniques, as shown here, can probe dynamic rearrangements within an enzyme’s active site during catalysis that cannot be resolved with any other current structural biological technique.

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Figure 1: Identification of single Trx catalytic events.
Figure 2: Force-dependent Trx catalysis.
Figure 3: Trx(P34H).
Figure 4: Structural model for force-dependent Trx catalysis.

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Acknowledgements

We thank D. Rodriguez-Larrea for thioredoxin purification and S. Posy for assistance with structural modelling. This work was supported by NIH grants to J.M.F., an NIH grant to B.J.B., a grant from the Swedish Society for Medical Research to A.H., and a grant from the Spanish Ministry of Science and Education to J.M.S.-R. F.G. is supported by an ISE Columbia University grant to J.M.F. and B.J.B. A.P.W. is supported by an NIH Medical Scientist Training Program grant to Columbia University.

Author Contributions A.P.W., R.P.-J. and J.M.F. designed the experiments. A.P.W. and R.P.-J. performed the experiments and analysed the data. K.A.W. designed the kinetic model and performed error analysis. F.G. and B.J.B. performed molecular dynamics simulations. A.H. provided TRX. J.M.S.-R. provided Trx and Trx(P34H). A.P.W., F.G., K.A.W., R.P.-J. and J.M.F. wrote the paper.

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Correspondence to Julio M. Fernandez.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

The file contains Supplementary Results, Supplementary Tables 1-4 and Supplementary Figures 1-10. This file includes the description of our theoretical model for estimating Δx12 in the Supplementary Results as well as all of the Supplementary Tables, Figures, and associated Legends referenced in the main text. (PDF 1441 kb)

Supplementary setup parameters 1

This file contains the setup parameters for the molecular dynamics (MD) simulations of the Trx:NF-kB complex. (TXT 3393 kb)

Supplementary setup parameters 2

This file contains the setup parameters for the molecular dynamics (MD) simulations of the unbound (apo) Trx enzyme. (TXT 2231 kb)

Supplementary setup parameters 3

This file contains the setup parameters for the molecular dynamics (MD) simulations of the Trx:Ref-1 complex (PDB: 1CQH). (TXT 2574 kb)

Supplementary MD simulations

The file contains overall parameters for the MD simulations (ZIP 2 kb)

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Wiita, A., Perez-Jimenez, R., Walther, K. et al. Probing the chemistry of thioredoxin catalysis with force. Nature 450, 124–127 (2007). https://doi.org/10.1038/nature06231

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