The role of arginine 120 of human prostaglandin endoperoxide H synthase-2 in the interaction with fatty acid substrates and inhibitors

Citation
Cj. Rieke et al., The role of arginine 120 of human prostaglandin endoperoxide H synthase-2 in the interaction with fatty acid substrates and inhibitors, J BIOL CHEM, 274(24), 1999, pp. 17109-17114
Citations number
38
Categorie Soggetti
Biochemistry & Biophysics
Journal title
JOURNAL OF BIOLOGICAL CHEMISTRY
ISSN journal
00219258 → ACNP
Volume
274
Issue
24
Year of publication
1999
Pages
17109 - 17114
Database
ISI
SICI code
0021-9258(19990611)274:24<17109:TROA1O>2.0.ZU;2-N
Abstract
Arg-120 is located near the mouth of the hydrophobic channel that forms the cyclooxygenase active site of prostaglandin endoperoxide H synthases (PGHS s)-1 and -2, Replacement of Arg-120 of ovine PGHS-1 with a glutamine increa ses the apparent K-m of PGHS-1 for arachidonate by 1,000-fold (Bhattacharyy a, D. K., Lecomte, M., Rieke, C. J., Garavito, R. M., and Smith, W. L. (199 6) J, Biol, Chem, 271, 2179-2184). This and other evidence indicate that th e guanido group of Arg-120 forms an ionic bond with the carboxylate group o f 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 simila r kinetic properties, but R120L hPGHS-2 catalyzes the oxygenation of arachi donate inefficiently. Our data indicate that the guanido group of Arg-120 o f hPGHS-2 interacts with arachidonate through a hydrogen bond rather than a n ionic bond and that this interaction is much less important for arachidon ate binding to PGHS-2 than to PGHS-1. The K-m values of PGHS-1 and -2 for a rachidonate are the same, and all but one of the core residues of the activ e 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 bindin g of arachidonate to PGHS-1 and -2 are quite different and that residues wi thin the hydrophobic cyclooxygenase channel must contribute more significan tly to arachidonate binding to PGHS-S than to PGHS-1. As observed previousl y with R120Q PGHS-1, flurbiprofen was an ineffective inhibitor of R120Q hPG HS-2, PGHS-2-specific inhibitors including NS398, DuP-697, and SC58125 had IC50 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 caus e time-dependent inhibition of R120Q hPGHS-2, whereas DuP-697 and SC58125 w ere time-dependent inhibitors. Thus, Arg-120 is important for the time-depe ndent inhibition of hPGHS-2 by NS398 but not by DuP-697 or SC58125.