Identification of human CYP2C19 residues that confer S-mephenytoin 4'-hydroxylation activity to CYP2C9.
Identification of human CYP2C19 residues that confer S-mephenytoin 4'-hydroxylation activity to CYP2C9.
复制标题
鉴定赋予 CYP2C9 S-美芬妥英 4-羟基化活性的人 CYP2C19 残基。
DOI：
10.1021/bi001678u
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发表时间：
2001
期刊：
影响因子：
2.9
通讯作者：
Goldstein,JA
中科院分区：
文献类型：
--
作者：
Tsao,CC;Wester,MR;Ghanayem,B;Coulter,SJ;Chanas,B;Johnson,EF;Goldstein,JA
CYP 2C 19对S-美芬妥英的4 '-羟基化具有选择性，而高度相似的CYP 2C 9对该底物几乎没有活性。为了鉴定决定人CYP 2C 19对S-美芬妥英4 '-羟基化特异性的关键氨基酸，我们通过用CYP 2C 19的那些取代含有各种拟定底物识别位点（SRS）的CYP 2C 9部分并通过定点诱变突变单个残基来构建嵌合体。只有包含SRS 1 - 4区域的嵌合体具有活性（野生型CYP 2C 19的30%），表明需要多个区域才能赋予S-美芬妥英特异性。诱变研究在将CYP 2C 9转化为S-美芬妥英4 '-羟化酶（野生型CYP 2C 19活性的6%）所需的蛋白质的三种拓扑组分中确定了6个残基。其中，只有位于SRS 1中螺旋B和C之间的I99 H差异反映了预测在血红素辅基上方形成的底物结合空腔中的侧链的变化。两个额外的取代，S220 P和P221 T位于螺旋F和G之间，但不靠近底物结合位点，加上螺旋I的N-末端部分的五个差异，赋予S-美芬妥英4 '-羟基化活性，其aKm与CYP 2C 19相似，但Kcat低3倍。螺旋I中的三个残基S286 N、V292 A和F295 L是S-美芬妥英4 '-羟基化活性所必需的。基于密切相关的酶CYP 2C 5的结构，这些残基在催化过程中不太可能直接接触底物，但其位置会影响底物结合位点残基的堆积，并可能影响酶中的底物进入通道。
CYP2C19 is selective for the 4‘-hydroxylation ofS-mephenytoin while the highly similar CYP2C9 has little activity toward this substrate. To identify critical amino acids determining the specificity of human CYP2C19 forS-mephenytoin 4‘-hydroxylation, we constructed chimeras by replacing portions of CYP2C9 containing various proposed substrate recognition sites (SRSs) with those of CYP2C19 and mutating individual residues by site-directed mutagenesis. Only a chimera containing regions encompassing SRSs 1−4 was active (30% of wild-type CYP2C19), indicating that multiple regions are necessary to confer specificity forS-mephenytoin. Mutagenesis studies identified six residues in three topological components of the proteins required to convert CYP2C9 to anS-mephenytoin 4‘-hydroxylase (6% of the activity of wild-type CYP2C19). Of these, only the I99H difference located in SRS 1 between helices B and C reflects a change in a side chain that is predicted to be in the substrate-binding cavity formed above the heme prosthetic group. Two additional substitutions, S220P and P221T residing between helices F and G but not in close proximity to the substrate binding site together with five differences in the N-terminal portion of helix I conferredS-mephenytoin 4‘-hydroxylation activity with aKMsimilar to that of CYP2C19 but a 3-fold lowerKcat. Three residues in helix I, S286N, V292A, and F295L, were essential forS-mephenytoin 4‘-hydroxylation activity. On the basis of the structure of the closely related enzyme CYP2C5, these residues are unlikely to directly contact the substrate during catalysis but are positioned to influence the packing of substrate binding site residues and likely substrate access channels in the enzyme.