Jiang Yongying, He Xiang, Ortiz de Montellano Paul R
Department of Pharmaceutical Chemistry, University of California, 600 16th Street, San Francisco, California 94143-2280, USA.
Biochemistry. 2006 Jan 17;45(2):533-42. doi: 10.1021/bi051840z.
Cytochromes P450cam and P450BM3 oxidize alpha- and beta-thujone into multiple products, including 7-hydroxy-alpha-(or beta-)thujone, 7,8-dehydro-alpha-(or beta-)thujone, 4-hydroxy-alpha-(or beta-)thujone, 2-hydroxy-alpha-(or beta-)thujone, 5-hydroxy-5-isopropyl-2-methyl-2-cyclohexen-1-one, 4,10-dehydrothujone, and carvacrol. Quantitative analysis of the 4-hydroxylated isomers and the ring-opened product indicates that the hydroxylation proceeds via a radical mechanism with a radical recombination rate ranging from 0.7 +/- 0.3 x 10(10) s(-1) to 12.5 +/- 3 x 10(10) s(-1) for the trapping of the carbon radical by the iron-bound hydroxyl radical equivalent. 7-[2H]-alpha-Thujone has been synthesized and used to amplify C-4 hydroxylation in situations where uninformative C-7 hydroxylation is the dominant reaction. The involvement of a carbon radical intermediate is confirmed by the observation of inversion of stereochemistry of the methyl-substituted C-4 carbon during the hydroxylation. With an L244A mutation that slightly increases the P450(cam) active-site volume, this inversion is observed in up to 40% of the C-4 hydroxylated products. The oxidation of alpha-thujone by human CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 occurs with up to 80% C-4 methyl inversion, in agreement with a dominant radical hydroxylation mechanism. Three minor desaturation products are produced, with at least one of them via a cationic pathway. The cation involved is proposed to form by electron abstraction from a radical intermediate. The absence of a solvent deuterium isotope effect on product distribution in the P450cam reaction precludes a significant role for the P450 ferric hydroperoxide intermediate in substrate hydroxylation. The results indicate that carbon hydroxylation is catalyzed exclusively by a P450 ferryl species via radical intermediates whose detailed properties are substrate- and enzyme-dependent.
细胞色素P450cam和P450BM3将α-和β-侧柏酮氧化为多种产物，包括7-羟基-α-（或β-）侧柏酮、7,8-脱氢-α-（或β-）侧柏酮、4-羟基-α-（或β-）侧柏酮、2-羟基-α-（或β-）侧柏酮、5-羟基-5-异丙基-2-甲基-2-环己烯-1-酮、4,10-脱氢侧柏酮和香芹酚。对4-羟基化异构体和开环产物的定量分析表明，羟基化通过自由基机制进行，铁结合的羟基自由基等效物捕获碳自由基的速率范围为0.7±0.3×10¹⁰ s⁻¹至12.5±3×10¹⁰ s⁻¹。已合成7-[2H]-α-侧柏酮，并用于在非信息性的C-7羟基化占主导反应的情况下增强C-4羟基化。在羟基化过程中观察到甲基取代的C-4碳的立体化学反转，证实了碳自由基中间体的参与。通过L244A突变略微增加P450(cam)活性位点体积后，在高达40%的C-4羟基化产物中观察到这种反转。人CYP1A2、CYP2C9、CYP2C19、CYP2D6、CYP2E1和CYP3A4对α-侧柏酮的氧化发生高达80%的C-4甲基反转，这与主要的自由基羟基化机制一致。产生了三种次要的去饱和产物，其中至少一种通过阳离子途径产生。所涉及的阳离子被认为是通过从自由基中间体夺取电子形成的。P450cam反应中产物分布不存在溶剂氘同位素效应，这排除了P450氢过氧化铁中间体在底物羟基化中起重要作用。结果表明，碳羟基化完全由P450铁氧物种通过自由基中间体催化，其详细性质取决于底物和酶。
