Hosny G, Roman L J, Mostafa M H, Masters B S
Department of Biochemistry, The University of Texas Health Science Center at San Antonio 78284-7760, USA.
Arch Biochem Biophys. 1999 Jun 15;366(2):199-206. doi: 10.1006/abbi.1999.1214.
Cytochromes P450 of the 4A family metabolize a variety of fatty acids, prostaglandins, and eicosanoids mainly at the terminal carbon (omega-hydroxylation) and, to a lesser extent, at the penultimate carbon [(omega-1)-hydroxylation]. In the present study, cytochrome P4504A5 (4A5) has been successfully expressed in Escherichia coli, with an average yield of enzyme of approximately 80 nmol/liter of cells. Spectroscopic characterization of the purified enzyme, using electron paramagnetic resonance and absolute and substrate-perturbed optical difference spectroscopy, showed that the heme of resting 4A5 is primarily low spin, but is converted primarily to high spin by substrate binding. The kcat and Km values for laurate omega-hydroxylation were 41 min-1 and 8.5 microM, respectively, in the absence of cytochrome b5, and 138 min-1 and 38 microM, respectively, in the presence of cytochrome b5. Hydroxylation of palmitate was dependent on the presence of cytochrome b5; kcat and Km values were 48 min-1 and 122 microM, respectively. Hydroxylation of arachidonic acid was barely detectable and was unchanged by the addition of cytochrome b5.
4A家族的细胞色素P450主要在末端碳原子处（ω-羟基化）代谢多种脂肪酸、前列腺素和类二十烷酸，在较小程度上也在倒数第二个碳原子处（ω-1-羟基化）进行代谢。在本研究中，细胞色素P4504A5（4A5）已在大肠杆菌中成功表达，每升细胞的酶平均产量约为80 nmol。使用电子顺磁共振以及绝对和底物扰动光学差光谱对纯化酶进行光谱表征，结果表明，静止状态的4A5的血红素主要为低自旋，但通过底物结合主要转变为高自旋。在不存在细胞色素b5的情况下，月桂酸ω-羟基化的kcat和Km值分别为41 min-1和8.5 μM，在存在细胞色素b5的情况下，分别为138 min-1和38 μM。棕榈酸的羟基化取决于细胞色素b5的存在；kcat和Km值分别为48 min-1和122 μM。花生四烯酸的羟基化几乎检测不到，并且添加细胞色素b5后也没有变化。
