Thioltransferase is a specific glutathionyl mixed disulfide oxidoreductase.
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.
Gravina
S A
SA
Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4965.
Mieyal
J J
JJ
D004220:Disulfides / Q000737:chemistry; D054477:Glutaredoxins; D005978:Glutathione / Q000378:metabolism*; D006454:Hemoglobins / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D010206:Papain / Q000378:metabolism; D011490:Protein Disulfide Reductase (Glutathione); D012710:Serum Albumin, Bovine / Q000378:metabolism; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013879:Thioredoxins / Q000378:metabolism
D004220:Disulfides; D054477:Glutaredoxins; D006454:Hemoglobins; D013438:Sulfhydryl Compounds; D012710:Serum Albumin, Bovine; D013879:Thioredoxins; D010088:Oxidoreductases; D011490:Protein Disulfide Reductase (Glutathione); D010206:Papain; D005978:Glutathione