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
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.