Oxidation of methyl and ethyl nitrosamines by cytochrome P450 2E1 and 2B1.
Oxidation of methyl and ethyl nitrosamines by cytochrome P450 2E1 and 2B1.
复制标题
DOI：
10.1021/bi301092c
复制
发表时间：
2012-12-18
期刊：
影响因子：
2.9
通讯作者：
Guengerich FP
中科院分区：
文献类型：
--
作者：
Chowdhury G;Calcutt MW;Nagy LD;Guengerich FP
细胞色素P450（P450）2 E1是氧化N-亚硝基二甲胺（N，N-dimethylnitrosamine，DMN）的主要酶。大鼠或人P450 2 E1氧化DMN生成HCHO的表观动力学氘同位素效应（KIE）≥ 8。在用大鼠肝微粒体进行的非竞争性分子间实验中，KIE没有减弱（DV 12.5，D（V/K）10.9，Northrop，D.B.命名法）。（1982）Methods Enzymol. 87，607-625），但与纯化的人P450 2 E1（DV 3.3，D（V/K）3.7），表明C-H键断裂对人P450 2 E1是部分限速的。对于N-亚硝基二乙胺（N，N-二乙基亚硝胺，DEN），内在KIE略低且不表达（例如，D（V/K）1.2）。在DMN和DEN的D（V/K）结果中也观察到了脱亚硝化反应产生的次要产物（CH 3 NH 2、CH 3CH 2NH 2和NO2−）的KIE的相同一般模式。氘代N-亚硝基-N-甲基，N-乙胺的实验表明，与甲基氧化相比，乙基的KIE较低，可以在单个分子内区分。P450 2 E1将DMN和DEN氧化成醛，然后氧化成羧酸。在酸形成中没有观察到动力学滞后;用载体醛进行的脉冲追踪实验仅显示与P450 2 E1结合的醛的有限平衡，指示进行性反应，如对P450 2A 6所报道的（Chowdhury，G.等人（2010）J.Biol.Chem.285，8031-8044）。这些相同的特征（没有HCO 2 H形成的滞后期，在脉冲追踪测定中缺乏平衡）也见于（大鼠）P450 2B 1，其对DMN氧化具有较低的催化效率和较大的活性位点。因此，二烷基亚硝胺氧化的持续合成能力似乎被许多P450所共享。
Cytochrome P450 (P450) 2E1 is the major enzyme that oxidizes N-nitrosodimethylamine (N,N-dimethylnitrosamine, DMN), a carcinogen and also a representative of some nitrosamines formed endogenously. Oxidation of DMN by rat or human P450 2E1 to HCHO showed a high apparent intrinsic kinetic deuterium isotope effect (KIE), ≥ 8. The KIE was not attenuated in non-competitive intermolecular experiments with rat liver microsomes (DV 12.5, D(V/K) 10.9, nomenclature of Northrop, D.B. (1982) Methods Enzymol. 87, 607–625) but was with purified human P450 2E1 (DV 3.3, D(V/K) 3.7), indicating that C-H bond breaking is partially rate-limiting with human P450 2E1. With N-nitrosodiethylamine (N,N-diethylnitrosamine, DEN), the intrinsic KIE was slightly lower and was not expressed (e.g., D(V/K) 1.2) in non-competitive intermolecular experiments. The same general pattern of KIEs was also seen in the D(V/K) results with DMN and DEN for the minor products resulting from the denitrosation reactions (CH3NH2, CH3CH2NH2, and NO2−). Experiments with deuterated N-nitroso-N-methyl,N-ethylamine demonstrated that the lower KIEs associated for ethyl compared to methyl oxidation could be distinguished within a single molecule. P450 2E1 oxidized DMN and DEN to aldehydes and then to the carboxylic acids. No kinetic lags were observed in acid formation; pulse-chase experiments with carrier aldehydes showed only limited equilibration with P450 2E1-bound aldehydes, indicative of processive reactions, as reported for P450 2A6 (Chowdhury, G. et al. (2010) J. Biol. Chem. 285, 8031–8044). These same features (no lag phase for HCO2H formation, lack of equilibration in pulse-chase assays) were also seen with (rat) P450 2B1, which has lower catalytic efficiency for DMN oxidation and a larger active site. Thus, the processivity of dialkylnitrosamine oxidation appears to be shared by a number of P450s.
登录
查看更多内容
影响因子：
2.9
作者：
GUENGERICH, FP;DANNAN, GA;KAMINSKY, LS
通讯作者：
KAMINSKY, LS
影响因子：
4.8
作者：
Bell, LC;Guengerich, FP
通讯作者：
Guengerich, FP
影响因子：
3.9
作者：
Guengerich FP;Sohl CD;Chowdhury G
通讯作者：
Chowdhury G
影响因子：
3.9
作者：
Guengerich, FP
通讯作者：
Guengerich, FP
影响因子：
4.7
作者：
ELESPURU, RK;SAAVEDRA, JE;LIJINSKY, W
通讯作者：
LIJINSKY, W