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. 2002 Jun 25;99(13):8579-84.
doi: 10.1073/pnas.122061399. Epub 2002 Jun 17.

Ebselen: a substrate for human thioredoxin reductase strongly stimulating its hydroperoxide reductase activity and a superfast thioredoxin oxidant

Rong Zhao  1 , Hiroyuki Masayasu, Arne Holmgren
Affiliations

Ebselen: a substrate for human thioredoxin reductase strongly stimulating its hydroperoxide reductase activity and a superfast thioredoxin oxidant

Rong Zhao et al. Proc Natl Acad Sci U S A. .

Abstract

Ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one], a seleno-organic compound with glutathione peroxidase-like activity is used in clinical trials against stroke. Human and bovine TrxR catalyzed the reduction of ebselen to ebselen selenol by NADPH with an apparent K(M)-value of 2.5 microM and a kcat of 588 min(-1). The addition of thioredoxin (Trx) stimulated the TrxR-catalyzed reduction of ebselen several-fold. This result was caused by a very fast oxidation of reduced Trx by ebselen with a rate constant in excess of 2 x 10(7) M(-1) s(-1). This rate is orders of magnitude faster than the reaction of dithiol Trx with insulin disulfides. Ebselen competed with disulfide substrates for reduction by Trx and, therefore, acted as an inhibitor of protein disulfide reduction by the Trx system. The inherent H2O2 reductase activity of mammalian TrxR dependent on its active-site selenocysteine residue was stimulated 10-fold by 2 microM ebselen and 25-fold in the additional presence of 5 microM Trx. Furthermore, the apparent K(M)-value of TrxR for H2O2 was lowered 25-fold to about 100 microM. Our results demonstrate that ebselen is a TrxR peroxidase which, in the presence of Trx, acted as a mimic of a peroxiredoxin. The activity with TrxR and oxidation of reduced Trx offer mechanistic explanations for the in vivo effects of ebselen as an antioxidant and anti-inflammatory agent. Our results demonstrate that the mechanism of action of ebselen may be predominantly via the Trx system rather than via glutathione.

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Figures

Figure 1
Figure 1
Reduction of ebselen by NADPH catalyzed by human TrxR. Concentrations of 10 μM (○, □) and 20 μM (●, ■) ebselen in 0.5 ml of 50 mM Tris⋅Cl/1 mM EDTA, pH 7.5 containing 100 μM NADPH was mixed with 10 nM (□, ■) and 50 nM (○, ●) human TrxR, and the A340 was followed against identical blank with enzyme but without ebselen.
Figure 2
Figure 2
Formation of ebselen selenol by reduction of 10 μM (●) and 20 μM (○) ebselen by 100 μM NADPH in 0.5 ml of 50 mM Tris⋅Cl/1 mM EDTA, pH 7.5 with 50 nM human TrxR. The A340 was followed against identical blank without the enzyme.
Scheme 1
Scheme 1
Figure 3
Figure 3
Lineweaver–Burk plot for ebselen as a substrate of mammalian TrxR. To cuvettes containing 0.5 ml of 50 mM Tris⋅Cl, 1 mM EDTA, pH 7.5, 100 μM NADPH, 1 mM H2O2, and 5 nM mammalian TrxR, 1–10 μM of ebselen was added. The A340 was followed against an identical blank without ebselen. The kcat was calculated by using the initial ΔA340 and the Δɛ of 6,200 M−1 cm−1 for the oxidation of NADPH.
Figure 4
Figure 4
Effect of human Trx on reduction of ebselen by TrxR. The oxidation of NADPH by 20 nM TrxR was recorded in the presence of no (■) or 5 μM (□) of hTrx-S2 in 0.5 ml of 50 mM Tris⋅Cl/1 mM EDTA, pH 7.5/100 μM NADPH. During the first 2 min, Trx-S2 was reduced to Trx-(SH)2. At the arrow, 10 μM ebselen was added to both cuvettes.
Figure 5
Figure 5
Oxidation of E. coli Trx-(SH)2 by ebselen determined by fluorescence spectroscopy. The sample contained 0.1 μM (1.2 μg/ml) of E. coli Trx-(SH)2 in N2-equilibrated 0.1 M potassium phosphate, 1 mM EDTA, pH 7.5. Fluorescence was excited at 290 nm and the emission spectrum from 300 to 500 nm was recorded. Then, 0.1 μM of ebselen was added and a new spectrum was recorded.
Figure 6
Figure 6
Reduction of H2O2 by human TrxR and the effect of ebselen and Trx. To cuvettes containing 50 mM Tris⋅Cl, 1 mM EDTA, pH 7.5, and 100 μM NADPH was added 0.5 mM H2O2 and 17 nM human TrxR (hTrxR) (●), 17 nM hTrxR plus 2 μM ebselen (▴), or 17 nM hTrxR plus 2 μM ebselen and 4.5 μM human Trx (hTrx) (■). The A340 was determined against a blank with 17 nM TrxR but without H2O2.
Figure 7
Figure 7
Effect of Trx and ebselen on reduction of H2O2 by TrxR. The same conditions as in Fig. 6 were used with only 17 nM TrxR (●) and the addition of 4.5 μM Trx (▵), followed by 0.5 μM ebselen (□) and, finally, ebselen to 5.5 μM (■).
Figure 8
Figure 8
Effect of different H2O2 concentrations on the activity of TrxR with ebselen. The reduction of H2O2 with 17 nM hTrxR and 2 μM ebselen (●) or with 17 nM hTrxR plus 4.5 μM hTrx and 2 μM ebselen (▵) was determined with the indicated concentrations of H2O2.
Scheme 2
Scheme 2
Scheme 3
Scheme 3
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