Role of Val(509) in time-dependent inhibition of human prostaglandin H synthase-2 cyclooxygenase activity by isoform-selective agents

Prostaglandin H synthase (PGHS), a hey 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(509) as a cyclooxygenase active site residue, that was not conserved in PGHS-1. Recombinant human PGHS-2 with Val(509) mutated to either Ile (the corresponding residue in PGHS-1), Ala, Glu, or Lys was expressed by transient transfection of COS-l cells to evaluate the effects of the mutations on cyclooxygenase activity and on inhibition by four agents reported to be selective for PGHS-2 (NS398, nimesulide, DuP697, and SC58125), All the recombinant proteins were of the expected mass, The mutants exhibited 45-210% of wild-type cyclooxygenase activity, with K-m values for arachidonate of 2.1-7.6 mu M (wild-type PGHS-2, 3.8 mu M), indicating that changes in position 509 had modest effects on cyclooxygenase catalysis, Each of the agents inhibited wild-type PGHS-2 in a Dime-dependent fashion, and all but nimesulide did the same for the V509A mutant, In contrast, the V509E and V509I PGHS-2 mutants, like recombinant human PGHS-1, did not show time-dependent inhibition with any of the agents, and the V509K mutant responded in a time-dependent manlier only to DuP697, Reversible inhibition was still observed with Val(509) mutants that did not show time-dependent inhibition, Thus, the side chain structure at position 509 markedly influenced the ability of PGHS-2 to undergo the time-dependent transition without removing inhibitor or substrate binding, These results indicate that Val(509) in PGHS-2 has a major role in the structural transition that underlies time-dependent inhibition by the isoform-selective agents.