Modeling of the active site of prostaglandin G/H synthase-2 (PGHS-2) onto 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 3 Arg and Ile 523 3 Val) and a double mutant (His 513 3 Arg,Ile 523 3 Val) containing both

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... was also constructed. The mutant enzyme forms were expressed in COS-7 cells, and their properties were compared with those of the normal isoforms using microsomal membranes. The mutated enzyme forms all had apparent K m values within 1.4-fold that of the wild type enzyme, and the specific activity of the mutants were within 2-fold of that of PGHS-1. DuP697, NS-398, DFU, and SC-58125 are selective PGHS-2 inhibitors that act as time-dependent inhibitors of PGHS-2 and rapidly reversible competitive inhibitors of PGHS-1. The single Ile 523 3 Val mutation increased the sensitivity to each of these selective inhibitors with most of the effect detected using instantaneous inhibition assays, except for DuP697, whose potency was further increased by preincubation with the enzyme. The double PGHS-1 His 513 3 Arg,Ile 523 3 Val mutant became more sensitive to inhibition by NS-398 and DFU than the single IV mutant, and time-dependent inhibition was observed. In contrast, the single HR mutation did not increase the sensitivity to inhibition by the selective PGHS-2 inhibitors. The potency of a selective PGHS-1 inhibitor, L-745,296, was decreased 5-and 13-fold in the HR and HR-IV mutants, respectively. All the results indicate that mutations of His 513 and Ile 523 residues of PGHS-1 can strongly increase sensitivity to selective PGHS-2 inhibition and restore time-dependent inhibition. They also suggest that the corresponding 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.