title: The catalytic mechanism of glutathione reductase as derived from x-ray diffraction analyses of reaction intermediates.
abstract: The mode of binding of NADPH and oxidized glutathione to the flavoenzyme glutathione reductase has been determined by x-ray crystallography. Furthermore, two intermediates of the reaction have been produced in the crystal and have been structurally elucidated. All these analyses were done at 0.3 nm resolution. The results allow the stereochemical description of the mechanism of the enzyme. The dinucleotide NADPH is bound in an extended conformation with the nicotinamide ring stacking onto the re-face of the flavin part of FAD, and adenine located at the protein surface. The binding of NADPH results in the 2-electron reduced form of the enzyme, EH2. This form has also been analyzed without any ligand bound. In EH2 the redoxactive disulfide bridge of the protein, which lies at the si-face of the flavin ring, is opened and the sulfur of Cys-58 moves by about 0.1 nm into a position where it can attack one of the sulfurs of the substrate oxidized glutathione. This interchange leads to a mixed glutathione-protein disulfide, which can be stabilized in crystals and has been analyzed. By selectively reacting Cys-58 with iodoacetamide the crystalline enzyme can be blocked in its EH2 state. The imidazole of His-467' is near to all sulfurs taking part in the disulfide bridge exchange and is therefore certainly crucial for catalysis. The crystallographic results establish that electrons flow from NADPH to the substrate GSSG via flavin and the redoxactive protein disulfide bridge. This is consistent with the scheme that has been postulated from biochemical, spectroscopic, and model studies.
mesh_terms: D001665:Binding Sites; D005980:Glutathione Reductase / Q000378:metabolism*; D008024:Ligands; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D014961:X-Ray Diffraction
chemical_list: D008024:Ligands; D013438:Sulfhydryl Compounds; D005980:Glutathione Reductase