Affinity isolation and characterization of cytochrome P450 102 (BM-3) from barbiturate-induced Bacillus megaterium.
Affinity isolation and characterization of cytochrome P450 102 (BM-3) from barbiturate-induced Bacillus megaterium.
复制标题
巴比妥诱导的巨大芽孢杆菌细胞色素 P450 102 (BM-3) 的亲和分离和表征。
DOI：
10.1006/abbi.1994.1148
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发表时间：
1994
影响因子：
3.9
通讯作者：
Galbraith,JA
中科院分区：
文献类型：
--
作者：
Black,SD;Linger,MH;Freck,LC;Kazemi,S;Galbraith,JA
细胞色素 P450 102 (BM-3) 是一种来自巨大芽孢杆菌的催化自给自足酶，目前被认为是哺乳动物微粒体 P450 单加氧酶系统的重要模型。我们开发了一种新颖的亲和方法，可在单个色谱步骤中纯化 P450 102，并研究了高度纯化的酶的光谱、催化、核苷酸结合和结晶特性。 megateriumATCC 14581 生长至高细胞密度，并且通过在腺苷-2'5'-二磷酸琼脂糖上从粗无细胞提取物中进行色谱法，快速且高产率地纯化 P450 102。细胞色素以显着的亲和力与柱结合，而 NADPH-细胞色素 P450 还原酶的结合明显较弱。色谱行为还表明，细胞色素与 NADP+ 型核苷酸的结合比任何其他细胞多肽更紧密。纯化的蛋白质是电泳均质的并且具有基本上理论含量的FAD、FMN和血红素。光谱显示了预期的血红素和黄素吸收带，并且表征了三种先前未描述的电荷转移型吸收。测定了氧化态、完全还原态和羰基亚铁态的摩尔消光系数；值得注意的是羰基亚铁态 (ϵ449nm- 143,500 M−1cm−1) 中的大索雷消光。最终制剂在多种底物的氧化中具有活性。在研究的 C14 烷基化合物中，十四烷基三甲基溴化铵表现出最高的 NADPH 底物依赖性氧化，其次是肉豆蔻酸酯和肉豆蔻醇；然而，肉豆蔻酸酯表现出最低的Km值。活性与 NADPH 氧化紧密相关 (>97%)。苯巴比妥、苯丙胺、可卡因、环己烷、甲醇、乙醇、视黄酸、苯甲酸盐、七氟丁酸盐和 7-乙氧基香豆素不是底物。正如预期的那样，NADP+滴定显示辅酶结合非常紧密，平均 Kd 为 580 nM。我们的 P450 102 制剂具有足够的纯度和稳定性，可以生长天然全酶晶体。
Cytochrome P450 102 (BM-3) is a catalytically self-sufficient enzyme fromBacillus megateriumthat is presently accepted as an important model of the mammalian microsomal P450 monooxygenase system. We have developed a novel affinity approach to purify P450 102 in a single chromatographic step and have studied the spectroscopic, catalytic, nucleotide binding, and crystallization properties of the highly purified enzyme.B. megateriumATCC 14581 was grown to high cell density, and P450 102 was purified rapidly and in high yield by chromatography on adenosine-2′5′-diphosphate agarose from crude cell-free extract. The cytochrome bound to the column with remarkable avidity, in contrast to the significantly weaker binding observed for NADPH-cytochrome P450 reductase. Chromatographic behavior also showed that the cytochrome bound NADP+-type nucleotides more tightly than any other cellular polypeptide. The purified protein was electrophoretically homogeneous and had essentially theoretical contents of FAD, FMN, and heme. Optical spectra showed the expected heme and flavin absorption bands, and three previously undescribed charge-transfer-type absorptions were characterized. Molar extinction coefficients in the oxidized, fully reduced, and ferrous carbonyl states have been determined; notable is the large soret extinction in the ferrous carbonyl state (ϵ449nm- 143,500 M−1cm−1). Final preparations were active in the oxidation of a wide variety of substrates. Of the C14alkyl compounds studied, tetradecyltrimethylammonium bromide showed the highest substrate-dependent oxidation of NADPH, followed by myristate and myristyl alcohol; however, myristate exhibited the lowestKmvalue. Activities were tightly coupled to NADPH oxidation (>97%). Phenobarbital, benzphetamine, cocaine, cyclohexane, methanol, ethanol, retinoic acid, benzoate, heptaflouro-butyrate, and 7-ethoxycoumarin were not substrates. NADP+titrations showed, as expected, that the coenzyme was bound very tightly, with an averageKdof 580 nM. Our preparations of P450 102 are of sufficient purity and stability that crystals of the native holoenzyme have been grown.