title: Role of Val509 in time-dependent inhibition of human prostaglandin H synthase-2 cyclooxygenase activity by isoform-selective agents.
abstract: 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 Val509 as a cyclooxygenase active site residue, that was not conserved in PGHS-1. Recombinant human PGHS-2 with Val509 mutated to either Ile (the corresponding residue in PGHS-1), Ala, Glu, or Lys was expressed by transient transfection of COS-1 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 Km values for arachidonate of 2.1-7.6 microM (wild-type PGHS-2, 3.8 microM), indicating that changes in position 509 had modest effects on cyclooxygenase catalysis. Each of the agents inhibited wild-type PGHS-2 in a time-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 manner only to DuP697. Reversible inhibition was still observed with Val509 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 Val509 in PGHS-2 has a major role in the structural transition that underlies time-dependent inhibition by the isoform-selective agents.
authors.affiliation: Department of Biology, University of Houston, Houston, Texas 77204, USA.
authors.forename: Q
authors.initials: Q
authors.lastname: Guo
authors.forename: L H
authors.initials: LH
authors.lastname: Wang
authors.forename: K H
authors.initials: KH
authors.lastname: Ruan
authors.forename: R J
authors.initials: RJ
authors.lastname: Kulmacz
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology*; D017931:DNA Primers; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011451:Prostaglandin-Endoperoxide Synthases / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D014633:Valine / Q000378:metabolism
chemical_list: D016861:Cyclooxygenase Inhibitors; D017931:DNA Primers; D007527:Isoenzymes; D011451:Prostaglandin-Endoperoxide Synthases; D014633:Valine