Identification of residues 99, 220, and 221 of human cytochrome P450 2C19 as key determinants of omeprazole activity.
Identification of residues 99, 220, and 221 of human cytochrome P450 2C19 as key determinants of omeprazole activity.
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鉴定人细胞色素 P450 2C19 的残基 99、220 和 221 作为奥美拉唑活性的关键决定因素。
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发表时间：
1996
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影响因子：
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通讯作者：
J. Goldstein
中科院分区：
文献类型：
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作者：
G. Ibeanu;B. Ghanayem;P. Linko;L. Li;L. G. Pederson;J. Goldstein
人P450 2C19对S-美苯妥因的4‘-羟化和奥美拉唑的5-羟化反应具有选择性，而结构上同源的P450 2C9对这些底物的活性较低。为了确定决定人P450 2C19特异性的关键氨基酸，我们构建了P450 2C9嵌合体，用P450 2C19取代了各种底物结合位点(SRS)，然后用定点突变取代了P450 2C19的单个残基，然后用定点突变取代了P450 2C9的单个残基。P450 2C19的339个NH2端氨基酸残基(SRS-1-SRS-4)和160-383个氨基酸(SRS-2-SRS-5)赋予了P450 2C9奥美拉唑5-羟基酶活性。在合同中，P450 2C19的COOH末端(残基340-490，包括SRS-5和SRS-6)、残基228-339(SRS-3和SRS-4)和残基292-383(SRS-4和SRS-5的一部分)的活性仅略有增加。单一突变Ile99-->；His使奥美拉唑5-羟基酶增加到P450 2C19的约51%。一个跨越P450 2C19 160-227残基的嵌合体也显示出奥美拉唑5-羟基酶活性，该活性被突变Ile99->；His显著增强。两个突变的组合，Ile99-->；His和Ser200-->；Pro，将P450 2C9转化为一种具有奥美拉唑5-羟基化的酶，这与P450/C19相似。Pro221-->；Thr突变增强了这一活性。残基99在SRS-1内，但氨基酸220和221在F-G环中，在任何已知的SRS之外。虽然含有P450 2C19的SRS-1-SRS-4和SRS-2-SRS-5的嵌合体显示出对该底物的活性，但这三个氨基酸的突变并未赋予P450 2C9显著的S-美苯妥因4‘-羟基酶活性。因此，我们的结果表明，氨基酸99、220和221是决定P450 2C19对奥美拉唑的特异性的关键残基。
Human P450 2C19 is selective for 4'-hydroxylation of S-mephenytoin and 5-hydroxylation of omeprazole, while the structurally homologous P450 2C9 has low activity toward these substrates. To identify the critical amino acids that determine the specificity of human amino acids that determine the specificity of human P450 2C19, we constructed chimeras of p450 2C9 replacing various proposed substrate binding sites (SRS) with those of P450 2C19 and then replaced individual residues of P450 2C19 and then replaced individual residues of P450 2C9 by site-directed mutagenesis. The 339 NH2-terminal amino acid residues (SRS-1-SRS-4) and amino acids 160-383 (SRS-2-SRS-5) of P450 2C19 conferred omeprazole 5-hydroxylase activity to P450 2C9. In contract, the COOH terminus of P450 2C19 (residues 340-490 including SRS-5 and SRS-6), residues 228-339 (SRS-3 and SRS-4) and residues 292-383 (part of SRS-4 and SRS-5) conferred only modest increases in activity. A single mutation Ile99 --> His increased omeprazole 5-hydroxylase to approximately 51% of that of P450 2C19. A chimera spanning residues 160-227 of P450 2C19 also exhibited omeprazole 5-hydroxylase activity which was dramatically enhanced by the mutation Ile99 --> His. A combination of two mutations, Ile99 --> His and Ser200 --> Pro, converted P450 2C9 to an enzyme with a turnover number of omeprazole 5-hyrdroxylation, which resembled that of P450 /c19. Mutation of Pro221 --> Thr enhanced this activity. Residue 99 is within SRS-1, but amino acids 220 and 221 are in the F-G loop and outside any known SRS. Mutation of these three amino acids did not confer significant S-mephenytoin 4'-hydroxylase activity to P450 2C9, although chimeras containing SRS-1-SRS-4 and SRS-2-SRS-5 of P450 2C19 exhibited activity toward this substrate. Our results thus indicate that amino acids 99, 220, and 221 are key residues that determine the specificity of P450 2C19 for omeprazole.