| 1. |
- Grahn, Elin, et al.
(författare)
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New crystal structures of human glutathione transferase A1-1 shed light on glutathione binding and the conformation of the C-terminal helix.
- 2006
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Ingår i: Acta Crystallogr D Biol Crystallogr. - 0907-4449. ; 62:Pt 2, s. 197-207
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Tidskriftsartikel (refereegranskat)abstract
- Human glutathione transferase A1-1 is a well studied enzyme, but despite a wealth of structural and biochemical data a number of aspects of its catalytic function are still poorly understood. Here, five new crystal structures of this enzyme are described that provide several insights. Firstly, the structure of a complex of the wild-type human enzyme with glutathione was determined for the first time at 2.0 angstroms resolution. This reveals that glutathione binds in the G site in a very similar fashion as the glutathione portion of substrate analogues in other structures and also that glutathione binding alone is sufficient to stabilize the C-terminal helix of the protein. Secondly, we have studied the complex with a decarboxylated glutathione conjugate that is known to dramatically decrease the activity of the enzyme. The T68E mutant of human glutathione transferase A1-1 recovers some of the activity that is lost with the decarboxylated glutathione, but our structures of this mutant show that none of the earlier explanations of this phenomenon are likely to be correct. Thirdly, and serendipitously, the apo structures also reveal the conformation of the crucial C-terminal region that is disordered in all previous apo structures. The C-terminal region can adopt an ordered helix-like structure even in the apo state, but shows a strong tendency to unwind. Different conformations of the C-terminal regions were observed in the apo states of the two monomers, which suggests that cooperativity could play a role in the activity of the enzyme.
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| 2. |
- Gustafsson, Ann, et al.
(författare)
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Role of the glutamyl alpha-carboxylate of the substrate glutathione in the catalytic mechanism of human glutathione transferase A1-1
- 2001
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 40:51, s. 15835-15845
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Tidskriftsartikel (refereegranskat)abstract
- The Glu alpha-carboxylate of glutathione contributes to the catalytic function of the glutathione transferases. The catalytic efficiency of human glutathione transferase A1-1 (GST A1-1) in the conjugation reaction with 1-chloro-2,4-dinitrobenzene is reduced 15 000-fold if the decarboxylated analogue of glutathione, dGSH (GABA-Cys-Gly), is used as an alternative thiol substrate. The decrease is partially due to an inability of the enzyme to promote ionization of dGSH. The pK(a) value of the thiol group of the natural substrate glutathione decreases from 9.2 to 6.7 upon binding to GST A1-1. However, the lack of the Glu alpha-carboxylate in dGSH raised the pK(a) value of the thiol in the enzymatic reaction to that of the nonenzymatic reaction. Furthermore, K(M)(dGSH) was 100-fold higher than K(M)(GSH). The active-site residue Thr68 forms a hydrogen bond to the Glu alpha-carboxylate of glutathione. Introduction of a carboxylate into GST A1-1 by a T68E mutation increased the catalytic efficiency with dGSH 10-fold and reduced the pK(a) value of the active site bound dGSH by approximately 1 pH unit. The altered pK(a) value is consistent with a catalytic mechanism where the carboxylate contributes to ionization of the glutathione thiol group. With Delta(5)-androstene-3,17-dione as substrate the efficiency of the enzyme is decreased 24 000-fold while with 4-nitrocinnamaldehyde (NCA) the decrease is less than 150-fold. In the latter reaction NCA accepts a proton and, unlike the other reactions studied, may not be dependent on the Glu alpha-carboxylate for deprotonation of the thiol group. An additional function of the Glu alpha-carboxylate may be productive orientation of glutathione within the active site.
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| 3. |
- Kurtovic, Sanela, et al.
(författare)
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Glutathione transferase activity with a novel substrate mimics the activation of the prodrug azathioprine
- 2008
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 375:2, s. 339-344
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Tidskriftsartikel (refereegranskat)abstract
- Azathioprine is a prodrug that is widely used clinically as an immunosuppressive agent. The pharmacological action of azathioprine is associated with the release of 6-mercaptopurine by a reaction involving glutathione. This biotransformation of azathioprine is catalyzed by glutathione transferases (GSTs). The nonenzymatic reaction with glutathione is minimal in comparison with the GST-catalyzed process, but azathioprine is still a slow substrate in comparison with the most effective GST substrates. Novel GSTs with higher catalytic efficiency toward azathioprine could be useful in novel therapeutic applications; therefore, directed evolution of GSTs for enhanced activities is desirable. However, screening for variants having higher catalytic activity with azathioprine is a time-consuming process due to the low activity with this substrate. A new chromogenic and faster substrate, 1-methyl-4-nitro-5-(4-nitrophenylthio)-1H-imidazole (NPTI), has been synthesized and characterized by assays with several GSTs. The novel substrate mimicked azathioprine in the reaction with glutathione catalyzed by alpha class GSTs and, therefore, is a valuable surrogate in the screening of large mutant libraries. NPTI may also find use in the elucidation of the exact mechanism of immunosuppression effected by azathioprine where there is evidence that the imidazole moiety of azathioprine, rather than 6-mercaptopurine, is involved.
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