Thioltransferase is a specific glutathionyl mixed-disulfide oxidoreductase

Abstract

To study the substrate specificity and mechanism of thioltransferase (TTase) catalysis, we have used 14C- and 35S-radiolabeled mixed disulfides of cysteine and glutathione (GSH) with various cysteine-containing proteins. These protein mixed disulfide substrates were incubated with glutathione, glutathione disulfide (GSSG) reductase, and NADPH in the presence or absence of thioltransferase. Glutathione-dependent reduction of protein mixed disulfides was monitored both by release of trichloroacetic acid soluble radiolabel and by formation of GSSG in an NADPH-linked spectrophotometric assay. GSH-dependent dethiolation of [35S]glutathione-papain mixed disulfide (papain-SSG) and the corresponding bovine serum albumin mixed disulfide (BSA-SSG) were catalyzed by thioltransferase (from human red blood cells) as shown by the radiolabel assay, and equivalent rates were measured by the spectrophotometric assay. Dethiolation of [35S]hemoglobin-glutathione mixed disulfide (Hb-SSG) was also catalyzed by TTase. In contrast, TTase did not catalyze GSH-dependent dethiolation of [14C]papain-SScysteine or [14C]BSA-SScysteine as measured by the radiolabel assay. [14C]Hb-SScysteine and Hb-SScysteamine also did not serve as substrates. In separate experiments, TTase from rat liver displayed analogous selectivity. Thus, thioltransferase (glutaredoxin) appears to be specific for glutathione-containing mixed disulfides. Apparent TTase catalysis of GSSG formation from the papain- and BSA-SScysteine mixed disulfides was observed by the spectrophotometric assay, but a lag phase occurred consistent with preenzymatic formation of GSScysteine which could serve as the actual TTase substrate. Two-substrate kinetic studies of TTase with GSH and GSScysteine gave patterns of parallel lines on double-reciprocal plots (1/V vs 1/[S]), consistent with a simple ping-pong mechanism involving a TTase-SSG intermediate.

Redox-based regulation of signal transduction: Principles, pitfalls, and promises

“…In addition to the monothiol reduction of glutathionyl disulfides, bacterial Grx also reduce low molecular weight disulfides, or disulfides in ribonucleotide reductase through a dithiol mechanism [83,99]. However for mammalian Grx, substrate specificity towards Sglutathionylated proteins has been demonstrated [101]. It is of interest to note that sulfiredoxin also was recently demonstrated to de-glutathionylate protein targets such as actin and protein tyrosine phosphatase 1B [102].…”

“…*1 In addition to the monothiol reduction of glutathionyl disulfides, bacterial Grx also reduces low molecular weight disulfides through a dithiol mechanism [83,99]. However mammalian GRX displays marked substrate specificity towards S-glutathionylated proteins [100,101]. Thus, the major target for Grx in mammalian cells in physiological setting is S-glutathione mixed disulfide (PSSG).…”

DHEA attenuates PDGF-induced phenotypic proliferation of vascular smooth muscle A7r5 cells through redox regulation

“…Glutaredoxin (GRX), a glutathione (GSH)-dependent oxidoreductase, catalyzes the reduction of protein disulfide via a disulfide exchange reaction [8].…”

Targeted disruption of the glutaredoxin 1 gene does not sensitize adult mice to tissue injury induced by ischemia/reperfusion and hyperoxia

“…Although both Trx1 and Grx1 exhibit activity in regeneration of oxidatively damaged proteins, further studies have shown that they have different substrate preferences [14,15]. Trx has been reported to preferentially reduce protein sulfenic acids as well as intra-and inter-protein disulfides.…”

“…In contrast, Grx is believed to play a unique role in the repair of oxidized protein thiols, specifically catalyzing the reduction of protein-SSG [3,15]. Since GSH is the most abundant non-protein thiol in cells (at concentrations from 0.5 to 20 mM) [19], the majority of protein mixed disulfides formed inside cells under oxidative stress is protein-SSG, and to a lesser extent intra-and inter-protein disulfides.…”