Probing the chemistry of thioredoxin catalysis with force

Abstract

Thioredoxins catalyze disulphide bond reduction in all living organisms. Single-molecule force-clamp spectroscopy has revealed that there are two alternative forms of the catalytic reaction: the first requires a reorientation of the disulphide bond in the substrate and the second involves an elongation of the disulphide bond in the substrate. 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.