Probing the chemistry of thioredoxin catalysis with force

Abstract

Thioredoxins are enzymes that catalyse disulphide bond reduction in all living organisms 1 . Although catalysis is thought to proceed through a substitution nucleophilic bimolecular (S N 2) reaction 1,2 , the role of the enzyme in modulating this chemical reaction is unknown. Here, using single-molecule force-clamp spectroscopy 3,4 , 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 disease 5,6 . 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.One of the principal challenges of understanding enzyme catalysis, a central problem in biology, is resolving the dynamics of enzyme-substrate interactions with sub-ångström resolution-the length scale at which chemistry occurs 7 . Although nuclear magnetic resonance (NMR) and X-ray crystallography determinations of protein structures can reach down to the sub-ångström level, they cannot yet provide dynamic information about enzyme

Crystal structure of human thioredoxin revealing an unraveled helix and exposed S‐nitrosation site

“…25,26 Most likely, this interface is generally used for specificity in interaction, and possibly also to generate correct alignment of target disulfide bonds with Cys 32, which single-molecule studies suggest requires rearrangement at the interface for formation of the intermolecular disulfide bond intermediate. 27,28 …”

Using multi-objective computational design to extend protein promiscuity

“…This enhancement is also evident in the lag-time values, which are significantly reduced in the case of V3 ( Figure 4C). Recent single-molecule force-clamp spectroscopy studies [37] have revealed two alternative forms of the catalytic mechanism of E. coli thioredoxin catalysis, the first requiring a re-orientation of the substrate disulfide bond and the second elongating the substrate disulfide bond. Given this complexity in the catalytic process, it does not seem possible at this stage to propose a reliable molecular interpretation of mutation effects …”

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“…A more recent AFM study achieved sub-angstrom spatial resolution sufficient to detect the rupture of single disulfide bonds in repeated arrays of thioredoxin. 57 …”