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

Abstract

Arg-120 is located near the mouth of the hydrophobic channel that forms the cyclooxygenase active site of prostaglandin endoperoxide H synthases (PGHSs)-1 and -2. Replacement of Arg-120 of ovine PGHS-1 with a glutamine increases the apparent K m of PGHS-1 for arachidonate by 1,000-fold (Bhattacharyya, D. K., Lecomte, M., Rieke, C. J., Garavito, R. M., and Smith, W. L. (1996) J. Biol. Chem. 271, 2179 -2184). This and other evidence indicate that the guanido group of Arg-120 forms an ionic bond with the carboxylate group of arachidonate and that this interaction is an important contributor to the overall strength of arachidonate binding to PGHS-1. In contrast, we report here that R120Q human PGHS-2 (hPGHS-2) and native hPGHS-2 have very similar kinetic properties, but R120L hPGHS-2 catalyzes the oxygenation of arachidonate inefficiently. Our data indicate that the guanido group of Arg-120 of hPGHS-2 interacts with arachidonate through a hydrogen bond rather than an ionic bond and that this interaction is much less important for arachidonate binding to PGHS-2 than to PGHS-1. The K m values of PGHS-1 and -2 for arachidonate are the same, and all but one of the core residues of the active sites of the two isozymes are identical. Thus, the results of our studies of Arg-120 of PGHS-1 and -2 imply that interactions involved in the binding of arachidonate to PGHS-1 and -2 are quite different and that residues within the hydrophobic cyclooxygenase channel must contribute more significantly to arachidonate binding to PGHS-2 than to PGHS-1. As observed previously with R120Q PGHS-1, flurbiprofen was an ineffective inhibitor of R120Q hPGHS-2. PGHS-2-specific inhibitors including NS398, DuP-697, and SC58125 had IC 50 values for the R120Q mutant that were up to 1,000-fold less than those observed for native hPGHS-2; thus, the positively charged guanido group of Arg-120 interferes with the binding of these compounds. NS398 did not cause time-dependent inhibition of R120Q hPGHS-2, whereas DuP-697 and SC58125 were time-dependent inhibitors. Thus, Arg-120 is important for the time-dependent inhibition of hPGHS-2 by NS398 but not by DuP-697 or SC58125.Prostaglandin endoperoxide H synthases (PGHSs) 1 catalyze the committed step in the formation of prostanoids, the conversion of arachidonic acid to prostaglandin (PG) H 2 (1). There are two PGHS isozymes. PGHS-1 is expressed constitutively in most tissues, and PGH 2 formed via PGHS-1 is involved in modulating and coordinating intercellular, intratissue responses to fluctuations in the levels of circulating hormones. PGHS-2 is expressed inducibly in response to cytokines (2-5), growth factors (6 -8), v-src (9), and tumor promoters (10), and products formed through the action of this isoform appear to play a role in cell growth and differentiation (11). Both PGHS-1 and PGHS-2 have similar kinetic and structural properties (1). Both isoforms catalyze two distinct reactions: (a) a cyclooxygenase reaction involving the bis-oxygenation of arachidonic acid to yield PGG 2 , and (b) a peroxidas...

Partnering between monomers of cyclooxygenase-2 homodimers

“…Adding a 10-or 100-fold excess of S-IBP did not change the properties of the melting curve; again, similar results were obtained with FBP (data not shown). These results were expected because neither FBP (18) (Fig. 3) nor S-IBP (data not shown) inhibits the R120Q huPGHS-2 homodimer.…”

“…Microsomal preparations of the R120Q huPGHS-2 homodimer have Ͼ90% of the COXspecific activity of the native huPGHS-2 homodimer when AA is used as the substrate (18). The specific activities of highly purified forms of R120Q͞R120Q huPGHS-2 and native͞R120Q huPGHS-2 were 87-90% of that of native͞native huPGHS-2 with AA and had comparable levels (80-110%) of POX activity (data not shown).…”

“…It is an effective time-dependent inhibitor of huPGHS-2 (23) but has been found not to inhibit R120Q huPGHS-2 even at concentrations of 1 mM (18). Nonetheless, FBP time-dependently inhibited the COX activities of both the native͞R120Q huPGHS-2 heterodimer and native͞native huPGHS-2 homodimer to similar extents (Ϸ29% and 18%, respectively) without inhibiting the R120Q͞R120Q homodimer (Fig.…”

“…The G533A mutation was chosen because these mutants have little COX activity with AA, linoleic acid, or ␣-linolenic acid but retain native POX activity (12,13). The R120Q monomer was used to generate heterodimers because the R120Q huPGHS-2 homodimer has COX kinetic properties similar to those of the native huPGHS-2 homodimer with AA, but, unlike native enzyme R120Q͞R120Q, huPGHS-2 is not inhibited effectively by FBP (18).…”

“…Arg-120 is found near the mouth of the COX active site of PGHSs and is involved in binding the carboxyl group of fatty acid substrates (11,16,17). An R120Q huPGHS-2 mutant can oxygenate AA but not eicosapentaenoic acid (EPA) (18); furthermore, FBP is Ϸ1,000 times less potent as an inhibitor of R120Q huPGHS-2 than native huPGHS-2.…”