CONVERSION OF PROSTAGLANDIN G H SYNTHASE-1 INTO AN ENZYME SENSITIVE TO PGHS-2-SELECTIVE INHIBITORS BY A DOUBLE HIS(513)-]ARG AND ILE(523)-]VAL MUTATION/

Modeling of the active site of prostaglandin G/H synthase-2 (PGHS-2) o nto PGHS-1 utilizing the known crystal structure of PGHS-1 shows that the only residues impinging directly on the active site that were not conserved in the two enzymes are His(513) and Ile(523) of PGHS-1 (Arg( 499) and Val(509) of PGHS-2). These residues of human PGHS-1 were each mutated to the corresponding PGHS-2 residues (His(513) --> Arg and Il e(523) --> Val) and a double mutant (His(513) --> Arg,Ile(523) --> Val ) containing both residues was also constructed. The mutant enzyme for ms were expressed in COS-7 cells, and their properties were compared w ith those of the normal isoforms using microsomal membranes. The mutat ed enzyme forms all had apparent K-m values within 1.4-fold that of th e wild type enzyme, and the specific activity of the mutants were with in 2-fold of that of PGHS-1. DuP697, NS-398, DFU, and SC-58125 are sel ective PGHS-2 inhibitors that act as time-dependent inhibitors of PGHS -2 and rapidly reversible competitive inhibitors of PGHS-1. The single Ile(523) --> Val mutation increased the sensitivity to each of these selective inhibitors with most of the effect detected using instantane ous inhibition assays, except for DuP697, whose potency was further in creased by preincubation with the enzyme. The double PGHS-1 His(513) - -> Arg,Ile(523) --> Val mutant became more sensitive to inhibition by NS-398 and DFU than the single IV mutant, and time-dependent inhibitio n was observed. In contrast, the single HR mutation did not increase t he sensitivity to inhibition by the selective PGHS-2 inhibitors. The p otency of a selective PGHS-1 inhibitor, L-745,296, was decreased 5- an d 13-fold in the HR and HR-IV mutants, respectively. All the results i ndicate that mutations of His(513) and Ile(523) residues of PGHS-1 can strongly increase sensitivity to selective PGHS-2 inhibition and rest ore time-dependent inhibition. They also suggest that the correspondin g Arg(499) and Val(509) residues of PGHS-2 are essential determinants in differentiating between the interaction of nonselective NSAIDs and selective PGHS-2 inhibitors and their mechanism of action.