The catalytic mechanism of the glutathione-dependent dehydroascorbate reductase activity of thioltransferase (glutaredoxin).
The catalytic mechanism of the glutathione (GSH)-dependent dehydroascorbic acid (DHA) reductase activity of recombinant pig liver thioltransferase (RPLTT) was investigated. RPLTT and the C25S mutant protein had equivalent specificity constants (kcat/Km) for both DHA and GSH. Iodoacetamide (IAM) inactivated the DHA reductase activities of RPLTT and C25S, confirming the essential role of cysteine in the reaction mechanism. When preincubated with DHA, RPLTT but not C25S was protected against IAM inactivation, suggesting that RPLTT has the ability to chemically reduce DHA forming ascorbic acid (AA) and the intramolecular disulfide form of the enzyme. Electrochemical detection of AA demonstrated the ability of both reduced RPLTT and C25S to chemically reduce DHA to AA in the absence of GSH. However, RPLTT had an initial rate of DHA reduction which was 4-fold greater than that of C25S, and after 10 min, RPLTT resulted in an AA concentration 11-fold greater than that of C25S. Isoelectric focusing analysis revealed that the product of reaction of reduced RPLTT but not C25S with DHA was consistent with the oxidized form of the enzyme. This result suggested that even though both RPLTT and the C25S mutant had equivalent specificity constants for DHA and GSH, they may have different catalytic mechanisms. On the basis of the experimental results, a catalytic mechanism for the DHA reductase activity of RPLTT is proposed. This is the first description of a catalytic mechanism of a glutathione:dehydroascorbate oxidoreductase (EC 1.8.5.1).
Washburn
M P
MP
Department of Biochemistry, Michigan State University, East Lansing 48824, USA.
Wells
W W
WW
D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism; D002384:Catalysis; D003545:Cysteine / Q000235:genetics; D003683:Dehydroascorbic Acid / Q000378:metabolism*; D004789:Enzyme Activation / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D054477:Glutaredoxins; D005978:Glutathione / Q000378:metabolism*; D007460:Iodoacetamide / Q000494:pharmacology; D007525:Isoelectric Focusing; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008956:Models, Chemical; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011490:Protein Disulfide Reductase (Glutathione); D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012694:Serine / Q000235:genetics; D013552:Swine
D004791:Enzyme Inhibitors; D054477:Glutaredoxins; D011994:Recombinant Proteins; D012694:Serine; D010088:Oxidoreductases; D011490:Protein Disulfide Reductase (Glutathione); C020666:glutathione dehydrogenase (ascorbate); D005978:Glutathione; D003545:Cysteine; D001205:Ascorbic Acid; D003683:Dehydroascorbic Acid; D007460:Iodoacetamide