Reactivity of the Human Thioltransferase (Glutaredoxin) C7S, C25S, C78S, C82S Mutant and NMR Solution Structure of Its Glutathionyl Mixed Disulfide Intermediate Reflect Catalytic Specificity
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- 作者：Yanwu Yang ; Shu-chuan Jao ; Sambasivarao Nanduri ; David W. Starke ; John J. Mieyal ; and Jun Qin
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：December 8, 1998
- 年：1998
- 卷：37
- 期：49
- 页码：17145 - 17156
- 全文大小：252K
- 年卷期：v.37,no.49(December 8, 1998)
- ISSN：1520-4995
文摘
Human thioltransferase (TTase) is a 12 kDa thiol-disulfide oxidoreductase that appears toplay a critical role in maintaining the redox environment of the cell. TTase acts as a potent and specificreducing agent for protein-S-S-glutathione mixed disulfides (protein-SSG) likely formed during oxidativestress or as redox intermediates in signal transduction pathways. Accordingly, the catalytic cycle ofthioltransferase itself involves a covalent glutathionyl enzyme disulfide intermediate (TTase-C22-SSG).To understand the molecular basis of TTase specificity for the glutathione moiety, we engineered aquadruple Cys to Ser mutant of human TTase (C7S, C25S, C78S, and C82S) which retains only theactive site cysteine residue (C22), and we solved its high-resolution NMR solution structure in the mixeddisulfide intermediate with glutathione (QM-TTase-SSG). This mutant which cannot form a C22-S-S-C25 intramolecular disulfide displays the same catalytic efficiency (Vmax/KM) and specificity for glutathionylmixed disulfide substrates as wild-type TTase, indicating that the Cys-25-SH moiety is not required forcatalysis or glutathionyl specificity. The structure of human thioltransferase is characterized by athioredoxin-like fold which comprises a four-stranded central 
-sheet flanked on each side by 
-helices.The disulfide-adducted glutathione in the TTase-SSG complex has an extended conformation and islocalized in a cleft near the protein surface encompassing the residues from helices-2,3, the active siteloop, and the loop connecting helix-3 and strand-3. Numerous van der Waals and electrostaticinteractions between the protein and the glutathione moiety are identified as contributing to stabilizationof the complex and confering the substrate specificity. Comparison of the human thioltransferase withother thiol-disulfide oxidoreductases reveals structural and functional differences.
-sheet flanked on each side by -helices.The disulfide-adducted glutathione in the TTase-SSG complex has an extended conformation and islocalized in a cleft near the protein surface encompassing the residues from helices-2,3, the active siteloop, and the loop connecting helix-3 and strand-3. Numerous van der Waals and electrostaticinteractions between the protein and the glutathione moiety are identified as contributing to stabilizationof the complex and confering the substrate specificity. Comparison of the human thioltransferase withother thiol-disulfide oxidoreductases reveals structural and functional differences.