Prostaglandin H synthase (PGHS), a key enzyme in prostanoid biosynthesis, exists as two isoforms. PGHS-1 is considered a basal enzyme; PGHS-2 is associated with inflammation and cell proliferation. A number of highly selective inhibitors for PGHS-2 cyclooxygenase activity are known. Inhibition by these agents involves an initial reversible binding, followed by a time-dependent transition to a much higher affinity enzyme-inhibitor complex, making these agents potent and poorly reversible PGHS-2 inhibitors. To investigate the PGHS-2 structural features that influence the time-dependent action of the selective inhibitors, we have constructed a three-dimensional model of human PGHS-2 by homologous modeling. Examination of the PGHS-2 model identified Val 1 catalyzes the first committed step in prostanoid biosynthesis, the bis-dioxygenation of arachidonic acid to form prostaglandin G 2 (1). Two isoforms of PGHS are known, with PGHS-1 generally ascribed housekeeping roles, whereas the strong induction of PGHS-2 by cytokines is believed to be a key part in inflammatory processes (2). Many cyclooxygenase inhibitors have been discovered; the most potent include agents, such as indomethacin, which trigger a time-dependent change in the protein once bound in the cyclooxygenase active site, thus achieving essentially irreversible inhibition without covalent modification of protein or agent (3-5). More recently, a set of time-dependent cyclooxygenase inhibitors with very high selectivity for PGHS-2 has been identified (6 -8). Little is known about the nature of the structural change(s) underlying noncovalent time-dependent cyclooxygenase inhibition of either isoform or about the protein structural features that lead to the remarkable specificity of the PGHS-2 inhibitors.We have constructed a three-dimensional model for human PGHS-2 based on the crystal structure of ovine PGHS-1 (9) and identified Val 509 in PGHS-2 as one of the few residues in the cyclooxygenase active site that is not conserved in PGHS-1. Recombinant human PGHS-2 was expressed with four Val 509 mutations to assess their effects on cyclooxygenase activity and on inhibition by agents specific for PGHS-2. Several of the Val 509 mutations led to a loss of the characteristic time-dependent action of the agents without a large perturbation of substrate or inhibitor binding. The results point to a role for Val 509 in the time-dependent structural transition, which makes these agents such potent and selective inhibitors of human PGHS-2 cyclooxygenase activity. EXPERIMENTAL PROCEDURESMaterials-Heme, dimethyl sulfoxide, and D-tryptophan were from Sigma; Tween 20 was from Pierce; arachidonate was from NuChek Preps, Inc.;[1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]arachidonate (55 mCi/mmol) was from Amersham Corp. Nimesulide was from Cayman Chemical Co. DuP697, SC58125, and NS398 were generous gifts from Drs. Chakk Ramesha (Roche Pharmaceuticals) and Paul J. Marshall (CIBA Pharmaceuticals).Homologous Modeling-The structural model for human PGHS-2 was built from th...

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