ARGININE-120 OF PROSTAGLANDIN G H SYNTHASE-1 IS REQUIRED FOR THE INHIBITION BY NONSTEROIDAL ANTIINFLAMMATORY DRUGS CONTAINING A CARBOXYLIC-ACID MOIETY/

The therapeutic action of nonsteroidal anti-inflammatory drugs (NSAIDs ) is exerted through the inhibition of prostaglandin G/H synthase (PGH S), which is expressed as two isoenzymes, termed PGHS-1 and PGHS-2. Fr om the crystal structure of sheep PGHS-1, it has been proposed that th e carboxylic acid group of flurbiprofen is located in a favorable posi tion for interacting with the arginine 120 residue of PGHS-1 (Picot, D ., Loll, P. J., and Garavito, R. M. (1994) Nature 367, 243-249). Mutat ion of this Arg(120) residue to Glu was performed and expressed in COS -7 cells using a vaccinia virus expression system. Comparison of micro somal enzyme preparations show that the mutation results in a 20-fold reduction in the specific activity of PGHS-1 and in a 100-fold increas e in the apparent K-m for arachidonic acid. Indomethacin, flurbiprofen , and ketoprofen, inhibitors of PGHS activity containing a free carbox ylic acid group, do not exhibit any inhibitory effects against the act ivity of PGHS-1(Arg(120) --> Glu). Diclofenac and meclofenamic acid, o ther NSAIDS containing a free carboxylic acid group, were 50-100-fold less potent inhibitors of the activity of the mutant as compared with the wild type PGHS. In contrast, the nonacid PGHS inhibitors, o-2-(4-f luorophenyl)-3-(4-methylsulfonyl)thiophene (DuP697) and a desbromo-sul fonamide analogue of DuP697 (L-746,483), were both more potent inhibit ors of PGHS-1(Arg(120) --> Glu) than of the wild type PGHS-1. Inhibiti on of PGHS-1(Arg(120) --> Glu) was time-dependent for diclofenac and t ime-independent for DuP697, as observed for the wild type enzyme, indi cating that the mutation does not alter the basic mechanism of inhibit ion. Aspirin is an acid NSAID that inhibits PGHS-1 through a unique co valent acetylation of the enzyme and also showed a reduced rate of ina ctivation of the mutated enzyme. These data provide biochemical eviden ce of the importance of the Arg(120) residue in PGHS-1 for interaction with arachidonic acid and NSAIDs containing a free carboxylic acid mo iety.