Hasler J A, Harlow G R, Szklarz G D, John G H, Kedzie K M, Burnett V L, He Y A, Kaminsky L S, Halpert J R
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson 85721.
Mol Pharmacol. 1994 Aug;46(2):338-45.
Eleven amino acid residues unique to dog cytochrome P450 (P450) 2B11, compared with rat 2B1 and 2B2, rabbit 2B4 and 2B5, and mouse 2B10, in the putative substrate recognition sites [J. Biol. Chem. 267:83-90 (1992)] were mutated to the residues found in 2B1 or 2B5. The mutants were expressed initially in COS cells and screened for activity toward androstenedione and 2,2',4,4',5,5'-hexachlorobiphenyl (245-HCB). P450 2B11 mutants V107I, M199L-N200E-V204R, V234I, A292L, Q473R, and I475S showed no differences from wild-type P450 2B11 in metabolite profiles with either substrate. Mutants V114I, D290I, and L363V exhibited altered androstenedione metabolite profiles and were expressed in Escherichia coli for further study with androstenedione, testosterone, 7-ethoxycoumarin, (R)- and (S)-warfarin, and 245-HCB. With V114I, hydroxylation of steroids and warfarin and 2-hydroxylation of 245-HCB were decreased, whereas 7-ethoxycoumarin O-dealkylation and 3-hydroxylation of 245-HCB were unaltered. For D290I, activities toward all substrates were decreased, except for 16 beta-hydroxylation of testosterone. The activity of L363V was increased 5-6-fold for 16 alpha-hydroxylation of androstenedione and testosterone but was decreased to 40-50% of wild-type activity with 7-ethoxycoumarin and warfarin and to 6-8% of control for 2-hydroxylation of 245-HCB. Alignment of P450 2B11 with P450 101 and super-imposition of the 11 mutated 2B11 residues on a P450 101 three-dimensional model suggest that only residues 114, 290, and 363 represent substrate contact residues, in excellent agreement with the experimental results. The data indicate the importance of the three residues 114, 290, and 363 in substrate specificity and regio- and stereoselectivity of P450 2B11 and also demonstrate that the effects of the mutations vary considerably with different substrates.
与大鼠2B1和2B2、兔2B4和2B5以及小鼠2B10相比，犬细胞色素P450（P450）2B11在假定的底物识别位点中具有11个独特的氨基酸残基[《生物化学杂志》267:83 - 90(1992)]，这些残基被突变为2B1或2B5中发现的残基。这些突变体最初在COS细胞中表达，并针对雄烯二酮和2,2',4,4',5,5'-六氯联苯（245 - HCB）的活性进行筛选。P450 2B11突变体V107I、M199L - N200E - V204R、V234I、A292L、Q473R和I475S在两种底物的代谢产物谱方面与野生型P450 2B11没有差异。突变体V114I、D290I和L363V表现出改变的雄烯二酮代谢产物谱，并在大肠杆菌中表达，以便进一步研究雄烯二酮、睾酮、7 - 乙氧基香豆素、（R） - 和（S） - 华法林以及245 - HCB。对于V114I，类固醇和华法林的羟基化以及245 - HCB的2 - 羟基化减少，而7 - 乙氧基香豆素O - 脱烷基化和245 - HCB的3 - 羟基化未改变。对于D290I，除睾酮的16β - 羟基化外，对所有底物的活性均降低。L363V对雄烯二酮和睾酮的16α - 羟基化活性增加了5 - 6倍，但对于7 - 乙氧基香豆素和华法林，其活性降至野生型活性的40 - 50%，对于245 - HCB的2 - 羟基化降至对照的6 - 8%。将P450 2B11与P450 101进行比对，并将11个突变的2B11残基叠加到P450 101三维模型上，结果表明只有114、290和363位残基代表底物接触残基，这与实验结果高度一致。数据表明114、290和363这三个残基在P450 2B11的底物特异性、区域和立体选择性中具有重要性，同时也证明了突变的影响因不同底物而有很大差异。
