| 1. |
- Balogh, Larissa M., et al.
(författare)
-
Structural Analysis of a Glutathione Transferase A1-1 Mutant Tailored for High Catalytic Efficiency with Toxic Alkenals
- 2009
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 48:32, s. 7698-7704
-
Tidskriftsartikel (refereegranskat)abstract
- The specificity of human glutathione transferase (GST) A1-1 is drastically altered to favor alkenal substrates in the GIMFhelix mutant designed to mimic first-sphere interactions utilized by GSTA4-4. This redesign serves as a model for improving our understanding of the structural determinants that contribute to the distinct specificities of alpha class GSTs. Herein we report the first crystal structures of GIMFhelix, both in complex with GSH and in apo form at 1.98 and 2.38 angstrom resolution. In contrast to the preorganized hydrophobic binding pocket that accommodates alkenals in GSTA4-4, GSTA1-1 includes a dynamic alpha 9 helix that undergoes a ligand-dependent localization to complete the active site. Comparisons of the GIMFhelix structures with previously reported structures show a striking similarity with the GSTA4-4 active site obtained within an essentially GSTA1-1 scaffold and reveal the 0 helix assumes a similar localized structure regardless of active site occupancy in a manner resembling that of GSTA4-4. However, Are cannot fully account for all the structural elements important in GSTA4-4 within the mutant's active site. The contribution of Phe10 to the Tyr212-Phe10-Phe220 network prevents complete C-terminal Closure and demonstrates that the presence of Phe10 within the context of a GSTA4-4-like active site may ultimately hinder Phe220, a key C-terminal residue, from effectively contributing to the active site. In total, these results illustrate the remaining structural differences presumably reflected in the previously reported catalytic efficiencies of GIMFhelix and GSTA4-4 and emphasize the F10P mutation as being necessary to completely accomplish the transformation to a highly specific GST from the more promiscuous GSTA1-1 enzyme.
|
|
| 2. |
|
|
| 3. |
|
|
| 4. |
- Hegazy, Usama M., et al.
(författare)
-
Modulating Catalytic Activity by Unnatural Amino Acid Residues in a GSH-Binding Loop of GST P1-1
- 2008
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 376:3, s. 811-826
-
Tidskriftsartikel (refereegranskat)abstract
- The loop following helix alpha2 in glutathione transferase P1-1 has two conserved residues, Cys48 and Tyr50, important for glutathione (GSH) binding and catalytic activity. Chemical modification of Cys48 thwarts the catalytic activity of the enzyme, and mutation of Tyr50 generally decreases the k(cat) value and the affinity for GSH in a differential manner. Cys48 and Tyr50 were targeted by site-specific mutations and chemical modifications in order to investigate how the alpha2 loop modulates GSH binding and catalysis. Mutation of Cys48 into Ala increased K(M)(GSH) 24-fold and decreased the binding energy of GSH by 1.5 kcal/mol. Furthermore, the protein stability against thermal inactivation and chemical denaturation decreased. The crystal structure of the Cys-free variant was determined, and its similarity to the wild-type structure suggests that the mutation of Cys48 increases the flexibility of the alpha2 loop rather than dislocating the GSH-interacting residues. On the other hand, replacement of Tyr50 with Cys, producing mutant Y50C, increased the Gibbs free energy of the catalyzed reaction by 4.8 kcal/mol, lowered the affinity for S-hexyl glutathione by 2.2 kcal/mol, and decreased the thermal stability. The targeted alkylation of Cys50 in Y50C increased the affinity for GSH and protein stability. Characterization of the most active alkylated variants, S-n-butyl-, S-n-pentyl-, and S-cyclobutylmethyl-Y50C, indicated that the affinity for GSH is restored by stabilizing the alpha2 loop through positioning of the key residue into the lock structure of the neighboring subunit. In addition, k(cat) can be further modulated by varying the structure of the key residue side chain, which impinges on the rate-limiting step of catalysis.
|
|
| 5. |
|
|
| 6. |
|
|
| 7. |
|
|
| 8. |
- Mandrika, Ilona, et al.
(författare)
-
QSAR of multiple mutated antibodies
- 2007
-
Ingår i: Journal of Molecular Recognition. - : Wiley. - 0952-3499 .- 1099-1352. ; 20:2, s. 97-102
-
Tidskriftsartikel (refereegranskat)abstract
- The aim of this study was to develop predictive quantitative structure-activity relationship (QSAR) modeling for antibody-peptide interactions. A small single chain antibody library was designed and manufactured around the murine anti-p24 (HIV-1) monoclonal antibody CB4-1 by use of statistical molecular design (SMD) principles and site directed mutagenesis, and its affinity for a p24 derived antigen was determined by fluorescence polarization. A satisfactory QSAR model (Q2 = 0.74, R2 = 0.88) was derived by correlating the affinity data to physicochemical property scales of the amino acids varied in the library. The model explains most of the antibody-antigen interactions of the studied set, and provides insights into the molecular mechanism involved in antigen binding.
|
|
| 9. |
|
|
| 10. |
|
|
