Wong E, Bayly C, Waterman H L, Riendeau D, Mancini J A
Department of Biochemistry and Molecular Biology, Merck Frosst, Kirkland, Quebec, H9H 3L1 Canada.
J Biol Chem. 1997 Apr 4;272(14):9280-6. doi: 10.1074/jbc.272.14.9280.
Modeling of the active site of prostaglandin G/H synthase-2 (PGHS-2) onto PGHS-1 utilizing the known crystal structure of PGHS-1 shows that the only residues impinging directly on the active site that were not conserved in the two enzymes are His513 and Ile523 of PGHS-1 (Arg499 and Val509 of PGHS-2). These residues of human PGHS-1 were each mutated to the corresponding PGHS-2 residues (His513 --> Arg and Ile523 --> Val) and a double mutant (His513 --> Arg,Ile523 --> Val) containing both residues was also constructed. The mutant enzyme forms were expressed in COS-7 cells, and their properties were compared with those of the normal isoforms using microsomal membranes. The mutated enzyme forms all had apparent Km values within 1.4-fold that of the wild type enzyme, and the specific activity of the mutants were within 2-fold of that of PGHS-1. DuP697, NS-398, DFU, and SC-58125 are selective PGHS-2 inhibitors that act as time-dependent inhibitors of PGHS-2 and rapidly reversible competitive inhibitors of PGHS-1. The single Ile523 --> Val mutation increased the sensitivity to each of these selective inhibitors with most of the effect detected using instantaneous inhibition assays, except for DuP697, whose potency was further increased by preincubation with the enzyme. The double PGHS-1 His513 --> Arg, Ile523 --> Val mutant became more sensitive to inhibition by NS-398 and DFU than the single IV mutant, and time-dependent inhibition was observed. In contrast, the single HR mutation did not increase the sensitivity to inhibition by the selective PGHS-2 inhibitors. The potency of a selective PGHS-1 inhibitor, L-745,296, was decreased 5- and 13-fold in the HR and HR-IV mutants, respectively. All the results indicate that mutations of His513 and Ile523 residues of PGHS-1 can strongly increase sensitivity to selective PGHS-2 inhibition and restore time-dependent inhibition. They also suggest that the corresponding Arg499 and Val509 residues of PGHS-2 are essential determinants in differentiating between the interaction of nonselective NSAIDs and selective PGHS-2 inhibitors and their mechanism of action.
利用前列腺素G/H合酶-1（PGHS-1）已知的晶体结构，将前列腺素G/H合酶-2（PGHS-2）的活性位点模拟到PGHS-1上，结果显示，在这两种酶中，直接作用于活性位点且不保守的唯一残基是PGHS-1的His513和Ile523（PGHS-2的Arg499和Val509）。将人PGHS-1的这些残基分别突变为相应的PGHS-2残基（His513→Arg和Ile523→Val），还构建了一个包含这两个残基的双突变体（His513→Arg，Ile523→Val）。突变酶形式在COS-7细胞中表达，并使用微粒体膜将其性质与正常同工型的性质进行比较。突变酶形式的表观Km值均在野生型酶的1.4倍以内，突变体的比活性在PGHS-1的2倍以内。DuP697、NS-398、DFU和SC-58125是选择性PGHS-2抑制剂，它们作为PGHS-2的时间依赖性抑制剂和PGHS-1的快速可逆竞争性抑制剂。单一的Ile523→Val突变增加了对这些选择性抑制剂中每一种的敏感性，除DuP697外，大部分效应在瞬时抑制试验中检测到，DuP697与酶预孵育后其效力进一步增加。双突变体PGHS-1 His513→Arg，Ile523→Val比单一IV突变体对NS-398和DFU的抑制更敏感，并且观察到时间依赖性抑制。相比之下，单一的HR突变并未增加对选择性PGHS-2抑制剂抑制的敏感性。选择性PGHS-1抑制剂L-745,296的效力在HR和HR-IV突变体中分别降低了5倍和13倍。所有结果表明，PGHS-1的His513和Ile523残基的突变可强烈增加对选择性PGHS-2抑制的敏感性并恢复时间依赖性抑制。它们还表明，PGHS-2相应的Arg499和Val509残基是区分非选择性非甾体抗炎药和选择性PGHS-2抑制剂相互作用及其作用机制的关键决定因素。
