Guengerich F P, Yun C H, Macdonald T L
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
J Biol Chem. 1996 Nov 1;271(44):27321-9. doi: 10.1074/jbc.271.44.27321.
Many enzymes catalyze N-dealkylations of alkylamines, including cytochrome P450 (P450) and peroxidase enzymes. Peroxidases, exemplified by horseradish peroxidase (HRP), are generally accepted to catalyze N-dealkylations via 1-electron transfer processes. Several lines of evidence also support a 1-electron mechanism for many P450 reactions, although this view has been questioned in light of reported trends for kinetic hydrogen isotope effects for N-demethylation with a series of 4-substituted N,N-dimethylanilines. No continuous trend for an increase of isotope effects with the electronic parameters of para-substitution was seen for the P450 2B1-catalyzed reactions in this study. The larger value seen with the 4-nitro derivative is consistent with a shift in mechanism due to either a reversible electron transfer step preceding deprotonation or to a hydrogen atom abstraction mechanism. With HRP, the trend is to lower isotope effects with para electron-withdrawing substituents, due to an apparent shift in rate-limiting steps. Biomimetic model high-valent porphyrins showed reduction rates with variously 4-substituted N,N-dialkylanilines that were consistent with a positively charged intermediate; such relationships were not seen for anisole O-demethylation with P450 2B1. In contrast to the case with the NADPH-supported P450 reactions, high deuterium isotope effects ( approximately 7) were seen in the N-dealkylations supported by the oxygen surrogate iodosylbenzene. With iodosylbenzene, colored aminium radicals were observed in the oxidations of aminopyrine, N,N-dimethyl-4-aminothioanisole, and 4-methoxy-N,N-dimethylaniline. With the latter compound, a substantial intermolecular deuterium isotope effect was observed for N-demethylation. In the N-dealkylation of N-ethyl,N-methylaniline by P450 2B1 (NADPH-supported), the ratio of N-demethylation to N-deethylation was 16. Although it is probably possible for P450s to catalyze amine N-dealkylations via hydrogen atom abstraction when such a course is electronically or sterically favored, we interpret the evidence to favor a 1-electron pathway with N,N-dialkylamines with P450 2B1 as well as HRP and several biomimetic models.
许多酶可催化烷基胺的N-脱烷基化反应，包括细胞色素P450（P450）和过氧化物酶。以辣根过氧化物酶（HRP）为代表的过氧化物酶，一般认为是通过单电子转移过程催化N-脱烷基化反应。尽管鉴于一系列4-取代的N,N-二甲基苯胺N-去甲基化反应中报道的动力学氢同位素效应趋势，这一观点受到了质疑，但几条证据也支持许多P450反应的单电子机制。在本研究中，P450 2B1催化的反应未观察到随着对位取代电子参数的增加同位素效应呈现连续趋势。4-硝基衍生物观察到的较大值与由于去质子化之前的可逆电子转移步骤或氢原子提取机制导致的机制转变一致。对于HRP，由于限速步骤的明显转变，对位吸电子取代基的趋势是降低同位素效应。仿生模型高价卟啉显示出与带正电荷中间体一致的、对各种4-取代的N,N-二烷基苯胺的还原速率；对于P450 2B1催化的苯甲醚O-去甲基化反应未观察到这种关系。与由NADPH支持的P450反应情况相反，在由氧替代物亚碘酰苯支持的N-脱烷基化反应中观察到高氘同位素效应（约为7）。使用亚碘酰苯时，在氨基比林、N,N-二甲基-4-氨基苯甲醚和4-甲氧基-N,N-二甲基苯胺的氧化反应中观察到有色铵自由基。对于后一种化合物，在N-去甲基化反应中观察到显著的分子间氘同位素效应。在P450 2B1（由NADPH支持）催化的N-乙基-N-甲基苯胺的N-脱烷基化反应中，N-去甲基化与N-去乙基化的比率为16。尽管当这样的过程在电子或空间上有利时，P450可能通过氢原子提取催化胺的N-脱烷基化反应，但我们解释这些证据支持P450 2B1以及HRP和几种仿生模型与N,N-二烷基胺反应时的单电子途径。
