Mancini J A, Riendeau D, Falgueyret J P, Vickers P J, O'Neill G P
Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Kirkland, Québec, Canada.
J Biol Chem. 1995 Dec 8;270(49):29372-7. doi: 10.1074/jbc.270.49.29372.
The therapeutic action of nonsteroidal anti-inflammatory drugs (NSAIDs) is exerted through the inhibition of prostaglandin G/H synthase (PGHS), which is expressed as two isoenzymes, termed PGHS-1 and PGHS-2. From the crystal structure of sheep PGHS-1, it has been proposed that the carboxylic acid group of flurbiprofen is located in a favorable position for interacting with the arginine 120 residue of PGHS-1 (Picot, D., Loll, P. J., and Garavito, R. M. (1994) Nature 367, 243-249). Mutation of this Arg120 residue to Glu was performed and expressed in COS-7 cells using a vaccinia virus expression system. Comparison of microsomal enzyme preparations show that the mutation results in a 20-fold reduction in the specific activity of PGHS-1 and in a 100-fold increase in the apparent Km for arachidonic acid. Indomethacin, flurbiprofen, and ketoprofen, inhibitors of PGHS activity containing a free carboxylic acid group, do not exhibit any inhibitory effects against the activity of PGHS-1(Arg120-->Glu). Diclofenac and meclofenamic acid, other NSAIDs containing a free carboxylic acid group, were 50-100-fold less potent inhibitors of the activity of the mutant as compared with the wild type PGHS. In contrast, the nonacid PGHS inhibitors, 5-bromo-2-(4-fluorophenyl)-3-(4-methylsulfonyl)thiophene (DuP697) and a desbromo-sulfonamide analogue of DuP697 (L-746,483), were both more potent inhibitors of PGHS-1(Arg120-->Glu) than of the wild tyupe PGHS-1. Inhibition of PGHS-1(Arg120-->Glu) was time-dependent for diclofenac and time-independent for DuP697, as observed for the wild type enzyme, indicating that the mutation does not alter the basic mechanism of inhibition. Aspirin is an acid NSAID that inhibits PGHS-1 through a unique covalent acetylation of the enzyme and also showed a reduced rate of inactivation of the mutated enzyme. These data provide biochemical evidence of the importance of the Arg120 residue in PGHS-1 for interaction with arachidonic acid and NSAIDs containing a free carboxylic acid moiety.
非甾体抗炎药（NSAIDs）的治疗作用是通过抑制前列腺素G/H合酶（PGHS）发挥的，该酶以两种同工酶形式存在，即PGHS-1和PGHS-2。根据绵羊PGHS-1的晶体结构，有人提出氟比洛芬的羧酸基团处于与PGHS-1的精氨酸120残基相互作用的有利位置（皮科特，D.，洛尔，P.J.，和加拉维托，R.M.（1994年）《自然》367，243 - 249）。将该精氨酸120残基突变为谷氨酸，并使用痘苗病毒表达系统在COS-7细胞中进行表达。微粒体酶制剂的比较表明，该突变导致PGHS-1的比活性降低20倍，花生四烯酸的表观Km增加100倍。吲哚美辛、氟比洛芬和酮洛芬是含有游离羧酸基团的PGHS活性抑制剂，对PGHS-1（精氨酸120→谷氨酸）的活性没有任何抑制作用。双氯芬酸和甲氯芬那酸是其他含有游离羧酸基团的NSAIDs，与野生型PGHS相比，它们对突变体活性的抑制效力低50 - 100倍。相比之下，非酸性PGHS抑制剂5-溴-2-（4-氟苯基）-3-（4-甲基磺酰基）噻吩（DuP697）和DuP697的去溴磺酰胺类似物（L-746,483）对PGHS-1（精氨酸120→谷氨酸）的抑制作用都比对野生型PGHS-1更强。如对野生型酶所观察到的那样，双氯芬酸对PGHS-1（精氨酸120→谷氨酸）的抑制作用是时间依赖性的，而DuP697的抑制作用是时间非依赖性的，这表明该突变不会改变抑制的基本机制。阿司匹林是一种酸性NSAID，它通过对该酶进行独特的共价乙酰化来抑制PGHS-1，并且对突变酶的失活速率也降低。这些数据提供了生化证据，证明PGHS-1中的精氨酸120残基对于与花生四烯酸和含有游离羧酸部分的NSAIDs相互作用的重要性。
