Reactivity of the Human Thioltransferase (Glutaredoxin) C7S, C25S, C78S, C82S Mutant and NMR Solution Structure of Its Glutathionyl Mixed Disulfide Intermediate Reflect Catalytic Specificity^,

Abstract

Human thioltransferase (TTase) is a 12 kDa thiol-disulfide oxidoreductase that appears to play a critical role in maintaining the redox environment of the cell. TTase acts as a potent and specific reducing agent for protein-S-S-glutathione mixed disulfides (protein-SSG) likely formed during oxidative stress or as redox intermediates in signal transduction pathways. Accordingly, the catalytic cycle of thioltransferase 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 a quadruple Cys to Ser mutant of human TTase (C7S, C25S, C78S, and C82S) which retains only the active site cysteine residue (C22), and we solved its high-resolution NMR solution structure in the mixed disulfide 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 glutathionyl mixed disulfide substrates as wild-type TTase, indicating that the Cys-25-SH moiety is not required for catalysis or glutathionyl specificity. The structure of human thioltransferase is characterized by a thioredoxin-like fold which comprises a four-stranded central beta-sheet flanked on each side by alpha-helices. The disulfide-adducted glutathione in the TTase-SSG complex has an extended conformation and is localized in a cleft near the protein surface encompassing the residues from helices-alpha2,alpha3, the active site loop, and the loop connecting helix-alpha3 and strand-beta3. Numerous van der Waals and electrostatic interactions between the protein and the glutathione moiety are identified as contributing to stabilization of the complex and confering the substrate specificity. Comparison of the human thioltransferase with other thiol-disulfide oxidoreductases reveals structural and functional differences.

Reversible Sequestration of Active Site Cysteines in a 2Fe-2S-bridged Dimer Provides a Mechanism for Glutaredoxin 2 Regulation in Human Mitochondria

“…For the E. coli enzymes, this covalent bond is the main interaction between glutathione and the protein, although GLRX3 has two additional polar contacts: the glutathione glutamate nitrogen to a threonine hydroxyl and the glutathione cysteine oxygen to a valine mainchain amide. Comparison of human GLRX1 (21) and GLRX2 indicates that the interaction between the glutathione glutamate carboxylate and the main-chain amide is preserved, but no side-chain interaction occurs in this area, and in GLRX1, the glutathione glycine carboxylate interacts only with the side chain of Arg-67. Perhaps the most important difference in ligand binding between the two human structures is that the Glu-Cys peptide bond of glutathione is flipped, with the peptide bond in the GLRX1 structure having an opposite orientation than in the other glutaredoxin structures.…”

“…This ligand binding behavior may also be the reason for the observed affinity of GLRX2 for GSH-Sepharose, a characteristic that distinguishes GLRX2 from other glutaredoxins (20). A search of the Protein Data Bank for glutaredoxins with a glutathione bound retrieved the following three structures: human glutaredoxin 1 (1B4Q) (21), Escherichia coli glutaredoxin 1 (1GRX) (22), and E. coli glutaredoxin 3 (3GRX) (23). All are NMR structures of a glutathioneprotein mixed disulfide with the N-terminal cysteine in the catalytic motif.…”

The Dithiol Glutaredoxins of African Trypanosomes Have Distinct Roles and Are Closely Linked to the Unique Trypanothione Metabolism

“…These residues are conserved in the trypanosomatid sequences. However, the Lys that precedes the active site motif as well as another charged or polar residue found to interact with the glycine carboxylate of GSH in various Grx structures (10,39,41,42) are replaced by hydrophobic residues. Interestingly, in E. coli Grx2, which has the highest activity of the three bacterial dithiol Grxs toward 2-ME-SSG, the Lys is replaced by a Tyr.…”

Comparative analysis of glutaredoxin domains from bacterial opportunistic pathogens

“…This might kill bacteria by inhibition of DNA synthesis and/or through increases in ROS toxicity. Structures have been published for several forms of human (Sun et al, 1998;Yang et al, 1998), plant (Rouhier et al, 2007;Li et al, 2010), budding yeast (Gibson et al, 2008;Discola et al, 2009) and Escherichia coli GLXRs (Iwema et al, 2009;Fladvad et al, 2005;Xia et al, 1992Xia et al, , 2001Bushweller et al, 1994;Sodano et al, 1991). However, it was unclear whether other bacterial GLXRs would adopt similar conformations.…”