Chaurasia C S, Alterman M A, Lu P, Hanzlik R P
Department of Medicinal Chemistry, University of Kansas, Lawrence 66045-2506.
Arch Biochem Biophys. 1995 Feb 20;317(1):161-9. doi: 10.1006/abbi.1995.1149.
The binding and hydroxylation of lauric acid by a genetically engineered and expressed fusion protein comprised of an N-truncated form of rat CYP4A1 linked to an N-truncated form of rat NADPH cytochrome P450 oxidoreductase (OR) (constructed by Fisher et al., (1992) Proc. Natl. Acad. Sci. USA 89, 10817-10822) has been characterized biochemically and compared to that shown by purified reconstituted rat CYP4A1 and liver microsomes from clofibrate-induced rats. In all systems lauric acid induced a Type I cytochrome P450 difference spectrum with Ks values in agreement with prior literature (range 10-18 microM). When provided with NADPH and oxygen but no other proteins or lipid, the fusion protein (called f4A1) catalyzed omega-hydroxylation of lauric acid with apparent Km and Vm of 3-4 microM and 4-5 nmol product/min/nmol P450 irrespective of buffer concentration or cation (NaPi or KPi, 25-200 mM); comparable values for reconstituted CYP4A1 and microsomes from clofibrate-induced rats are 9 microM and 34 min-1 and 5 microM and 10 min-1, respectively. (omega-1)-Hydroxylation of lauric acid was barely detectable (omega/(omega-1) = 135) with f4A1 or with reconstituted CYP4A1, but it accounted for up to 50% of total products formed by microsomes from clofibrate-induced rats. When added to the f4A1 system, OR stimulated hydroxylation up to fivefold at a OR:f4A1 ratio of 5:1; additionally, (omega-1)-hydroxylation was routinely observed as a minor process (< 4% of total product) in this system. These effects were also independent of buffer concentration. In contrast addition of cytochrome b5 (b5) caused a small (25%) decrease in omega-hydroxylation, while added phospholipid had no effect. However, the combination of OR, b5, and lipid stimulated turnover approximately 10-fold compared to f4A1 alone, and 11-hydroxylauric acid was regularly formed as a minor (3-4% of total) product. These observations indicate that the fusion protein f4A1 is functionally equivalent to reconstituted CYP4A1 with respect to binding and hydroxylation of lauric acid and suggest that it can be used as an alternative to reconstituted systems for structure-function and mechanistic studies of fatty acid omega-hydroxylation.
一种由大鼠CYP4A1的N端截短形式与大鼠NADPH细胞色素P450氧化还原酶（OR）的N端截短形式连接而成的基因工程表达融合蛋白（由Fisher等人于1992年构建，《美国国家科学院院刊》89卷，10817 - 10822页）对月桂酸的结合和羟基化作用已通过生化方法进行了表征，并与纯化的重组大鼠CYP4A1以及氯贝丁酯诱导的大鼠肝脏微粒体的作用进行了比较。在所有系统中，月桂酸均诱导出I型细胞色素P450差异光谱，其Ks值与先前文献一致（范围为10 - 18 microM）。当提供NADPH和氧气但不添加其他蛋白质或脂质时，融合蛋白（称为f4A1）催化月桂酸的ω-羟基化反应，其表观Km和Vm分别为3 - 4 microM和4 - 5 nmol产物/分钟/nmol P450，与缓冲液浓度或阳离子（NaPi或KPi，25 - 200 mM）无关；重组CYP4A1和氯贝丁酯诱导的大鼠微粒体的相应值分别为9 microM和34分钟^-1以及5 microM和10分钟^-1。f4A1或重组CYP4A1对月桂酸的（ω-1）-羟基化反应几乎检测不到（ω/（ω-1） = 135），但氯贝丁酯诱导的大鼠微粒体形成的总产物中，（ω-1）-羟基化产物占比高达50%。当添加到f4A1系统中时，OR以5:1的OR:f4A ratio刺激羟基化反应高达五倍；此外，在该系统中，（ω-1）-羟基化反应通常作为一个次要过程被观察到（占总产物的< 4%）。这些效应也与缓冲液浓度无关。相比之下，添加细胞色素b5（b5）会使ω-羟基化反应略有下降（25%），而添加磷脂则没有影响。然而，与单独的f4A1相比，OR、b5和脂质的组合使周转率提高了约10倍，并且11-羟基月桂酸经常作为次要产物（占总量的3 - 4%）形成。这些观察结果表明，融合蛋白f4A1在月桂酸的结合和羟基化方面在功能上等同于重组CYP4A1，并表明它可作为重组系统的替代物，用于脂肪酸ω-羟基化的结构-功能和机制研究。
