Letter

Nature 450, 124-127 (1 November 2007) | doi:10.1038/nature06231; Received 21 May 2007; Accepted 7 September 2007

Probing the chemistry of thioredoxin catalysis with force

Arun P. Wiita1,2, Raul Perez-Jimenez1, Kirstin A. Walther1,3, Frauke Gräter4, B. J. Berne4, Arne Holmgren5, Jose M. Sanchez-Ruiz6 & Julio M. Fernandez1

  1. Department of Biological Sciences,
  2. Graduate Program in Neurobiology and Behavior,
  3. Department of Physics,
  4. Department of Chemistry, Columbia University, New York, New York 10027, USA
  5. Medical Nobel Institute for Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77, Stockholm, Sweden
  6. Facultad de Ciencias, Departamento de Quimica Fisica, Universidad de Granada, 18071, Granada, Spain

Correspondence to: Julio M. Fernandez1 Correspondence and requests for materials should be addressed to J.M.F. (Email: jfernandez@columbia.edu).

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 plusminus 0.09 Å (plusminus s.e.m.), and the second elongating the substrate disulphide bond by 0.17 plusminus 0.02 Å (plusminus 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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