Boddupalli S S, Estabrook R W, Peterson J A
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235-9038.
J Biol Chem. 1990 Mar 15;265(8):4233-9.
Cytochrome P-450BM-3 is a catalytically self-sufficient enzyme which monooxygenates saturated and unsaturated fatty acids, alcohols, and amides. The protein has two domains: one which contains heme and is P-450-like and the other which contains FAD and FMN and is P-450 reductase-like. Both domains are on a single polypeptide chain. Utilizing a plasmid containing the gene encoding P-450BM-3, we have transformed the Escherichia coli strain DH5 alpha. This clone overexpresses P-450BM-3 to make approximately 20% of the soluble protein of this organism under optimal conditions. P-450BM-3 can be purified to homogeneity from the soluble fraction of the protein of these cells with a recovery of 50% making this cell line an excellent source of this important enzyme. Purified preparations of P-450BM-3 hydroxylate palmitic acid at a rate of 1600 mol/min/mol of heme at 25 degrees C. The stoichiometry of NADPH to oxygen utilized was 1 for all conditions; however, the ratio of oxygen or NADPH utilized per molecule of fatty acid substrate metabolized was different for different homologs of saturated fatty acids, when low concentrations (less than 100 microM) of substrate were used. Lauric and myristic acids were metabolized to two hydroxylated products, irrespective of the initial concentration of fatty acid in the reaction mixture, and the ratio of oxygen consumed to fatty acid hydroxylated was 1. High concentrations of palmitic acid (greater than 200 microM) led to the formation of three polar metabolites and a stoichiometry of 1:1 was observed for oxygen and palmitic acid utilization. These results indicate that a single hydroxyl group was inserted into each of these molecules. Lower concentrations (less than 50 microM) of palmitic acid were metabolized to additional polar metabolites, and the ratio of oxygen consumed to fatty acid substrate consumed approximated 3:1. These results can be explained best by a hypothesis that the initial hydroxylated compounds, which accumulate during the oxidation of palmitic acid by P-450BM-3, can be further oxidized by this enzyme to polyhydroxy- or hydroxy-ketone products.
细胞色素P-450BM-3是一种具有催化自足性的酶，可将饱和脂肪酸和不饱和脂肪酸、醇类及酰胺进行单加氧反应。该蛋白质有两个结构域：一个含有血红素，类似P-450；另一个含有黄素腺嘌呤二核苷酸（FAD）和黄素单核苷酸（FMN），类似P-450还原酶。两个结构域位于一条单一的多肽链上。利用一个含有编码P-450BM-3基因的质粒，我们对大肠杆菌菌株DH5α进行了转化。在最佳条件下，该克隆过量表达P-450BM-3，使其占该生物体可溶性蛋白质的约20%。P-450BM-3可从这些细胞蛋白质的可溶性部分纯化至同质，回收率为50%，这使得该细胞系成为这种重要酶的优良来源。纯化后的P-450BM-3制剂在25℃下以每摩尔血红素1600摩尔/分钟的速率使棕榈酸羟基化。在所有条件下，所利用的烟酰胺腺嘌呤二核苷酸磷酸（NADPH）与氧气的化学计量比均为1；然而，当使用低浓度（小于100微摩尔）的底物时，每代谢一分子脂肪酸底物所利用的氧气或NADPH的比例对于不同的饱和脂肪酸同系物是不同的。月桂酸和肉豆蔻酸被代谢为两种羟基化产物，与反应混合物中脂肪酸的初始浓度无关，消耗的氧气与羟基化脂肪酸的比例为1。高浓度的棕榈酸（大于200微摩尔）导致形成三种极性代谢产物，并且观察到氧气与棕榈酸利用的化学计量比为1:1。这些结果表明在这些分子的每一个中都插入了一个单一的羟基。较低浓度（小于50微摩尔）的棕榈酸被代谢为其他极性代谢产物，消耗的氧气与消耗的脂肪酸底物的比例约为3:1。这些结果最好用一个假说来解释，即在P-450BM-3氧化棕榈酸过程中积累的初始羟基化化合物可被该酶进一步氧化为多羟基或羟基酮产物。
