Conversion of Prostaglandin G/H Synthase-1 into an Enzyme Sensitive to PGHS-2-selective Inhibitors by a Double His513→ Arg and Ile523→ Val Mutation

Abstract

Modeling of the active site of prostaglandin G/H synthase-2 (PGHS-2) onto PGHS-1 utilizing the known crystal structure of PGHS-1 shows that the only residues impinging directly on the active site that were not conserved in the two enzymes are His 513 and Ile 523 of PGHS-1 (Arg 499 and Val 509 of PGHS-2). These residues of human PGHS-1 were each mutated to the corresponding PGHS-2 residues (His 513 3 Arg and Ile 523 3 Val) and a double mutant (His 513 3 Arg,Ile 523 3 Val) containing both residues was also constructed. The mutant enzyme forms were expressed in COS-7 cells, and their properties were compared with those of the normal isoforms using microsomal membranes. The mutated enzyme forms all had apparent K m values within 1.4-fold that of the wild type enzyme, and the specific activity of the mutants were within 2-fold of that of PGHS-1. DuP697, NS-398, DFU, and SC-58125 are selective PGHS-2 inhibitors that act as time-dependent inhibitors of PGHS-2 and rapidly reversible competitive inhibitors of PGHS-1. The single Ile 523 3 Val mutation increased the sensitivity to each of these selective inhibitors with most of the effect detected using instantaneous inhibition assays, except for DuP697, whose potency was further increased by preincubation with the enzyme. The double PGHS-1 His 513 3 Arg,Ile 523 3 Val mutant became more sensitive to inhibition by NS-398 and DFU than the single IV mutant, and time-dependent inhibition was observed. In contrast, the single HR mutation did not increase the sensitivity to inhibition by the selective PGHS-2 inhibitors. The potency of a selective PGHS-1 inhibitor, L-745,296, was decreased 5-and 13-fold in the HR and HR-IV mutants, respectively. All the results indicate that mutations of His 513 and Ile 523 residues of PGHS-1 can strongly increase sensitivity to selective PGHS-2 inhibition and restore time-dependent inhibition. They also suggest that the corresponding Arg 499 and Val 509 residues of PGHS-2 are essential determinants in differentiating between the interaction of nonselective NSAIDs and selective PGHS-2 inhibitors and their mechanism of action.Prostaglandins are derived from arachidonic acid and act as mediators of pain, fever, and other inflammatory responses (1). Prostaglandin G/H synthase (PGHS)1 converts arachidonic acid into prostaglandin G 2 by the addition of molecular oxygen (a cyclooxygenase step) and then catalyzes the conversion of prostaglandin G 2 to prostaglandin H 2 by a peroxidase reaction (2, 3). Prostaglandin H 2 is the precursor to the formation of all prostaglandins, thromboxane, and prostacyclin. Nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin and indomethacin, abrogate prostaglandin synthesis through inhibition of the cyclooxygenase reaction of PGHS (4).A second isoform of PGHS has been discovered (PGHS-2) that is induced in inflammatory situations in response to cytokines or growth factors (5-10). This has lead to the development of selective PGHS-2 inhibitors, which have demonstrated that inhibition of PGHS-2 alone is suffici...

The Role of Arginine 120 of Human Prostaglandin Endoperoxide H Synthase-2 in the Interaction with Fatty Acid Substrates and Inhibitors

“…3). Other results that are consistent with this interpretation include the following: (a) there is a large decrease in cyclooxygenase catalytic efficiency associated with the mutation of Arg-120 to glutamate (34,37) or leucine in PGHS-2; (b) the crystal structure of mouse apo-PGHS-2 with arachidonate bound in the cyclooxygenase site suggests that the carboxylate group is in close proximity to Arg-120 (35) …”

Developmental expression of functional cyclooxygenases in zebrafish

“…1). The volume of the arachidonate-binding channel of mammalian COX-1 enzymes is determined by Ile-523, His-513, and Ile-434 (17)(18)(19). These residues are substituted by Val-523, Arg-513, and Val-434 in human COX-2, which opens access to a side pocket in the arachidonate-binding channel for selective COX-2 inhibitors.…”

Structural and Functional Basis of Cyclooxygenase Inhibition

“…Substitution of the COX-1 residues in COX-2 at the active site mouth has no effect on the selectivity of compounds 2 and 3 for COX-2, indicating that the single Val-523 difference between COX-1 and COX-2 is sufficient to confer COX-2 selectivity for these inhibitors. The replacement of Ile-523 in COX-1 with valine increases the affinity of the enzyme for COX-2 selective inhibitors 98 and the substitution of His-513 in COX-1 for arginine in COX-2 changes the chemical environment of the side pocket. 31 In combination, the I523V and H513R reverse mutants in COX-1 are much more sensitive to inhibition by diaryl heterocycles.…”

“…31 In combination, the I523V and H513R reverse mutants in COX-1 are much more sensitive to inhibition by diaryl heterocycles. 98 The first crystal structure of human COX-2 (hCOX-2) shows that there is an overall difference in the size and shape of the COX-2 active site compared to that of COX-1. 30 The nearly 25% larger active site of COX-2 is accounted for by the single Val-523 substitution (Ile in COX-1) in the active site and by the Arg-513 and Val-434 substitutions (His-513 and Ile-434 in COX-1) in the secondary shell.…”