Fang X, Halpert J R
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson 85721, USA.
Drug Metab Dispos. 1996 Nov;24(11):1282-5.
The ability of dithionite, an inexpensive reducing agent routinely used to produce the ferrous-carbonyl form of P450, to support P450BM-3-catalyzed hydroxylation of palmitate was studied. The hydroxylation products in the presence of dithionite were 15, 14, and 13-hydroxyhexadecanoate, with relative distributions similar to those observed with NADPH. The hydroxylation reaction was carried out in two separate steps, anaerobic reduction and subsequent oxidation of P450BM-3 by oxygen bubbling. The reduction step was much slower than the oxidation step, thus limiting the overall rate of hydroxylation. Upon addition of dithionite, the reductase domain of P450BM-3 seemed to be reduced before significant reduction of the heme domain occurred. The discovery of new reducing agents for P450-catalyzed reaction raises the possibility of replacing NADPH in specialty chemical hydroxylation catalyzed by P450s, especially catalytically self-sufficient P450s, such as P450BM-3 or recombinant fusion proteins of P450 covalently linked to a reductase.
研究了连二亚硫酸盐（一种常用于制备P450亚铁羰基形式的廉价还原剂）支持P450BM-3催化棕榈酸酯羟基化的能力。在连二亚硫酸盐存在下的羟基化产物为15-、14-和13-羟基十六烷酸酯，其相对分布与用NADPH观察到的相似。羟基化反应分两个独立步骤进行，即厌氧还原以及随后通过鼓泡氧气对P450BM-3进行氧化。还原步骤比氧化步骤慢得多，因此限制了羟基化的总体速率。加入连二亚硫酸盐后，P450BM-3的还原酶结构域似乎在血红素结构域显著还原之前就已被还原。发现用于P450催化反应的新还原剂增加了在由P450催化的特种化学品羟基化反应中取代NADPH的可能性，特别是催化自足的P450，如P450BM-3或与还原酶共价连接的P450重组融合蛋白。
