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. Access through

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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. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Biological Sciences,, Arun P. Wiita, Raul

Perez-Jimenez, Kirstin A. Walther & Julio M. Fernandez * Graduate Program in Neurobiology and Behavior,, Arun P. Wiita * Department of Physics,, Kirstin A. Walther * Department of

Chemistry, Columbia University, New York, New York 10027, USA, Frauke Gräter & B. J. Berne * Department of Medical Biochemistry and Biophysics, Medical Nobel Institute for Biochemistry,

Karolinska Institutet, SE-171 77, Stockholm, Sweden, Arne Holmgren * Departamento de Quimica Fisica, Facultad de Ciencias, Universidad de Granada, 18071, Granada, Spain, Jose M. Sanchez-Ruiz

Authors * Arun P. Wiita View author publications You can also search for this author inPubMed Google Scholar * Raul Perez-Jimenez View author publications You can also search for this

author inPubMed Google Scholar * Kirstin A. Walther View author publications You can also search for this author inPubMed Google Scholar * Frauke Gräter View author publications You can also

search for this author inPubMed Google Scholar * B. J. Berne View author publications You can also search for this author inPubMed Google Scholar * Arne Holmgren View author publications

You can also search for this author inPubMed Google Scholar * Jose M. Sanchez-Ruiz View author publications You can also search for this author inPubMed Google Scholar * Julio M. Fernandez

View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Julio M. Fernandez. ETHICS DECLARATIONS COMPETING INTERESTS 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) RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE 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 Download citation *

Received: 21 May 2007 * Accepted: 07 September 2007 * Issue Date: 01 November 2007 * DOI: https://doi.org/10.1038/nature06231 SHARE THIS ARTICLE Anyone you share the following link with will

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