Arginine 120 of Prostaglandin G/H Synthase-1 Is Required for the Inhibition by Nonsteroidal Anti-inflammatory Drugs Containing a Carboxylic Acid Moiety

• Published in: The Journal of biological chemistry 1995-12, Vol.270 (49), p.29372-29377
• Main Authors: Mancini, J A, Riendeau, D, Falgueyret, J P, Vickers, P J, O'Neill, G P
• Format: Article
• Language: English
• Subjects: Animals; Anti-Inflammatory Agents, Non-Steroidal - pharmacology; Arachidonic Acid - metabolism; Arginine; Base Sequence; Cells, Cultured; Cyclooxygenase Inhibitors - pharmacology; Molecular Sequence Data; Mutation; Oxidation-Reduction; Prostaglandin-Endoperoxide Synthases - chemistry; Structure-Activity Relationship
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.270.49.29372

The therapeutic action of nonsteroidal anti-inflammatory drugs (NSAIDs) is exerted through the inhibition of prostaglandin G/H synthase (PGHS), which is expressed as two isoenzymes, termed PGHS-1 and PGHS-2. From the crystal structure of sheep PGHS-1, it has been proposed that the carboxylic acid group of flurbiprofen is located in a favorable position for interacting with the arginine 120 residue of PGHS-1 (Picot, D., Loll, P. J., and Garavito, R. M.(1994) Nature 367, 243-249). Mutation of this Arg residue to Glu was performed and expressed in COS-7 cells using a vaccinia virus expression system. Comparison of microsomal enzyme preparations show that the mutation results in a 20-fold reduction in the specific activity of PGHS-1 and in a 100-fold increase in the apparent K for arachidonic acid. Indomethacin, flurbiprofen, and ketoprofen, inhibitors of PGHS activity containing a free carboxylic acid group, do not exhibit any inhibitory effects against the activity of PGHS-1(Arg Glu). Diclofenac and meclofenamic acid, other 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, 5-bromo-2-(4-fluorophenyl)-3-(4-methylsulfonyl)thiophene (DuP697) and a desbromo-sulfonamide analogue of DuP697 (L-746,483), were both more potent inhibitors of PGHS-1(Arg Glu) than of the wild type PGHS-1. Inhibition of PGHS-1(Arg Glu) was time-dependent for diclofenac and time-independent for DuP697, as observed for the wild type enzyme, indicating that the mutation does not alter the basic mechanism of inhibition. Aspirin is an acid NSAID that inhibits PGHS-1 through a unique covalent acetylation of the enzyme and also showed a reduced rate of inactivation of the mutated enzyme. These data provide biochemical evidence of the importance of the Arg residue in PGHS-1 for interaction with arachidonic acid and NSAIDs containing a free carboxylic acid moiety.