THE 3-DIMENSIONAL STRUCTURE OF CYS-47-MODIFIED MOUSE-LIVER GLUTATHIONE-S-TRANSFERASE P1-1 - CARBOXYMETHYLATION DRAMATICALLY DECREASES THE AFFINITY FOR GLUTATHIONE AND IS ASSOCIATED WITH A LOSS OF ELECTRON-DENSITY IN THE ALPHA-B-3(10)B REGION

The three-dimensional structure of mouse liver glutathione S-transfera se P1-1 carboxymethylated at Cys-47 and its complex with S-(p-nitroben zyl)glutathione have been determined by x-ray diffraction analysis, Th e structure of the modified enzyme described here is the first structu ral report for a P-i class glutathione S-transferase with no glutathio ne, glutathione S-conjugate, or inhibitor bound, It shows that part of the active site area, which includes helix alpha B and helix 3(10)B, is disordered. However, the environment of Tyr-7, an essential residue for the catalytic reaction, remains unchanged, The position of the su lfur atom of glutathione is occupied in the ligand-free enzyme by a wa ter molecule that is at H-bond distance from Tyr-7, We do not find any structural evidence for a tyrosinate form, and therefore our results suggest that Tyr-7 is not acting as a general base abstracting the pro ton from the thiol group of glutathione, The binding of the inhibitor S-(p-nitrobenzyl)glutathione to the carboxymethylated enzyme results i n a partial restructuring of the disordered area. The modification of Cys-47 sterically hinders structural organization of this region, and although it does not prevent glutathione binding, it significantly red uces the affinity, A detailed kinetic study of the modified enzyme ind icates that the carboxymethylation increases the K-m for glutathione b y 3 orders of magnitude, although the enzyme can function efficiently under saturating conditions.