The Catalytic Mechanism of the Glutathione-Dependent Dehydroascorbate Reductase Activity of Thioltransferase (Glutaredoxin)
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- 作者：Michael P. Washburn and William W. Wells
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：January 5, 1999
- 年：1999
- 卷：38
- 期：1
- 页码：268 - 274
- 全文大小：91K
- 年卷期：v.38,no.1(January 5, 1999)
- ISSN：1520-4995
文摘
The catalytic mechanism of the glutathione (GSH)-dependent dehydroascorbic acid (DHA)reductase activity of recombinant pig liver thioltransferase (RPLTT) was investigated. RPLTT and theC25S 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 ofcysteine in the reaction mechanism. When preincubated with DHA, RPLTT but not C25S was protectedagainst IAM inactivation, suggesting that RPLTT has the ability to chemically reduce DHA formingascorbic acid (AA) and the intramolecular disulfide form of the enzyme. Electrochemical detection ofAA demonstrated the ability of both reduced RPLTT and C25S to chemically reduce DHA to AA in theabsence of GSH. However, RPLTT had an initial rate of DHA reduction which was 4-fold greater thanthat of C25S, and after 10 min, RPLTT resulted in an AA concentration 11-fold greater than that ofC25S. Isoelectric focusing analysis revealed that the product of reaction of reduced RPLTT but not C25Swith DHA was consistent with the oxidized form of the enzyme. This result suggested that even thoughboth RPLTT and the C25S mutant had equivalent specificity constants for DHA and GSH, they mayhave different catalytic mechanisms. On the basis of the experimental results, a catalytic mechanism forthe DHA reductase activity of RPLTT is proposed. This is the first description of a catalytic mechanismof a glutathione:dehydroascorbate oxidoreductase (EC 1.8.5.1).