Rieke C J, Mulichak A M, Garavito R M, Smith W L
Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824, USA.
J Biol Chem. 1999 Jun 11;274(24):17109-14. doi: 10.1074/jbc.274.24.17109.
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 Km 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 Km 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 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 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.
精氨酸 - 120位于疏水通道口附近，该疏水通道构成前列腺素内过氧化物H合酶（PGHSs）-1和 - 2的环氧化酶活性位点。用谷氨酰胺取代绵羊PGHS - 1的精氨酸 - 120会使PGHS - 1对花生四烯酸的表观Km增加1000倍（Bhattacharyya, D. K., Lecomte, M., Rieke, C. J., Garavito, R. M., and Smith, W. L. (1996) J. Biol. Chem. 271, 2179 - 2184）。这一现象及其他证据表明，精氨酸 - 120的胍基与花生四烯酸的羧基形成离子键，且这种相互作用是花生四烯酸与PGHS - 1结合总体强度的重要贡献因素。相比之下，我们在此报告，R120Q人PGHS - 2（hPGHS - 2）和天然hPGHS - 2具有非常相似的动力学性质，但R120L hPGHS - 2催化花生四烯酸的氧化效率低下。我们的数据表明，hPGHS - 2的精氨酸 - 120的胍基通过氢键而非离子键与花生四烯酸相互作用，且这种相互作用对花生四烯酸与PGHS - 2的结合而言，远不如其与PGHS - 1的结合重要。PGHS - 1和 - 2对花生四烯酸的Km值相同，且这两种同工酶活性位点的核心残基除一个之外全部相同。因此，我们对PGHS - 1和 - 2的精氨酸 - 120的研究结果表明，花生四烯酸与PGHS - 1和 - 2结合所涉及的相互作用差异很大，且疏水环氧化酶通道内的残基对花生四烯酸与PGHS - 2的结合贡献比对PGHS - 1的结合贡献更为显著。如先前在R120Q PGHS - 1中观察到的那样，氟比洛芬是R120Q hPGHS - 2的无效抑制剂。包括NS398、DuP - 697和SC58125在内的PGHS - 2特异性抑制剂对R120Q突变体的IC50值比对天然hPGHS - 2观察到的IC50值低至1000倍；因此，精氨酸 - 120带正电荷的胍基会干扰这些化合物的结合。NS398不会引起R120Q hPGHS - 2的时间依赖性抑制，而DuP - 697和SC58125是时间依赖性抑制剂。因此，精氨酸 - 120对NS398引起的hPGHS - 2时间依赖性抑制很重要，但对DuP - 697或SC58125引起的抑制不重要。
