Thioltransferase in human red blood cells: kinetics and equilibrium.
Thioltransferase from human red blood cells (HRBC TTase), coupled to GSSG reductase, catalyzed glutathione (GSH)-dependent reduction of prototype substrates hydroxyethyl disulfide (HEDS) and sodium S-sulfocysteine as well as of other homo- and heterodisulfides, including the protein mixed disulfide albumin-S-S-cysteine. Whereas apparent KM values for the substrates varied over more than a 20-fold range, the Vmax values agreed quite closely, usually within less than a factor of 2, suggesting that initial interaction of oxidized substrate with enzyme is not rate determining. HRBC TTase was inactivated by iodoacetamide (IAA), and this was prevented by pretreatment with disulfides. The pH dependence of IAA inactivation gave a remarkably low apparent pKa of 3.5, which was independent of ionic strength (0.05-2 M). At pH 6, one radiolabeled carboxyamidomethyl moiety was bound to the enzyme after treatment with [14C]IAA. This unusual thiol reactivity suggests that the active-site cysteine moiety of the TTase may be involved in a hydrogen bond with a carboxylate moiety. In contrast, the pH dependence for GSH-dependent TTase catalysis of disulfide reduction displayed an inflection point near pH 8.0, also suggesting that the initial reaction of oxidized substrate with the active-site thiol is not involved in rate determination. Two substrate kinetic studies of HRBC TTase and rat liver TTase (e.g., [GSH] and [HEDS] varied independently) gave patterns of intersecting lines on double-reciprocal plots (1/v vs 1/S), indicating a sequential mechanism for the TTase reactions, rather than a ping-pong mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)
Mieyal
J J
JJ
Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.
Starke
D W
DW
Gravina
S A
SA
Hocevar
B A
BA
D000818:Animals; D002384:Catalysis; D004220:Disulfides; D004912:Erythrocytes / Q000201:enzymology*; D054477:Glutaredoxins; D005980:Glutathione Reductase / Q000097:blood; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000097:blood*; D011490:Protein Disulfide Reductase (Glutathione); D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds
D004220:Disulfides; D054477:Glutaredoxins; D013438:Sulfhydryl Compounds; D010088:Oxidoreductases; D005980:Glutathione Reductase; D011490:Protein Disulfide Reductase (Glutathione)