Masuda Kazufumi, Tamagake Keietsu, Okuda Yukie, Torigoe Fumihiro, Tsuzuki Daisuke, Isobe Takashi, Hichiya Hiroyuki, Hanioka Nobumitsu, Yamamoto Shigeo, Narimatsu Shizuo
Laboratory of Pharmaceutical Physical Chemistry, Faculty of Pharmaceutical Sciences, Okayama University, Okayama, Japan.
Chirality. 2005 Jan;17(1):37-43. doi: 10.1002/chir.20092.
The functional roles of phenylalanine at position 120 in drug oxidation by cytochrome P450 2D6 (CYP2D6) were examined using a yeast cell expression system and bufuralol (BF) enantiomers as a chiral substrate. Two mutated cDNAs, one encoding a CYP2D6 mutant having alanine instead of Phe-120 (F120A) and another encoding a mutant having alanine instead of Glu-222 (E222A), were prepared by site-directed mutagenesis and transformed into yeast cells via pGYRI vectors. The enantiomeric BF 1''-hydroxylase activities of the mutants were compared with those of the wild type. When enantiomeric BF 1''-hydroxylase activities at a substrate concentration of 100 microM were compared, the CYP2D6 wild type showed substrate enantioselectivity of (R-BF >> S-BF) and the F120A mutant exhibited substrate enantioselectivity of (R-BF < or = S-BF), whereas the product diastereoselectivity of (1''R-OH-BF << 1''-S-OH-BF) was similar between the wild type and the mutant. The activities of the other mutant (E222A) were much lower than those of the wild type and the F120A mutant, while its substrate enantioselectivity and product diastereoselectivity were the same as those of the wild type. The kinetics demonstrated that apparent K(m) values were similar among the recombinant enzymes, and V(max) values clearly reflected the selectivity described above. These results indicate that Phe-120 has a key role in the enantioselective BF 1''-hydroxylation by CYP2D6.
利用酵母细胞表达系统和作为手性底物的布呋洛尔（BF）对映体，研究了细胞色素P450 2D6（CYP2D6）中120位苯丙氨酸在药物氧化中的功能作用。通过定点诱变制备了两个突变cDNA，一个编码用丙氨酸取代苯丙氨酸-120（F120A）的CYP2D6突变体，另一个编码用丙氨酸取代谷氨酸-222（E222A）的突变体，并通过pGYRI载体将其转化到酵母细胞中。将突变体的对映体BF 1''-羟化酶活性与野生型进行比较。当比较底物浓度为100 microM时的对映体BF 1''-羟化酶活性时，CYP2D6野生型表现出（R-BF >> S-BF）的底物对映选择性，而F120A突变体表现出（R-BF ≤ S-BF）的底物对映选择性，而野生型和突变体之间（1''R-OH-BF << 1''-S-OH-BF）的产物非对映选择性相似。另一个突变体（E222A）的活性远低于野生型和F120A突变体，但其底物对映选择性和产物非对映选择性与野生型相同。动力学表明，重组酶之间的表观K(m)值相似，V(max)值清楚地反映了上述选择性。这些结果表明，苯丙氨酸-120在CYP2D6对映选择性BF 1''-羟化中起关键作用。
