Marshall P J, Kulmacz R J
Department of Biological Chemistry, University of Illinois, Chicago 60612.
Arch Biochem Biophys. 1988 Oct;266(1):162-70. doi: 10.1016/0003-9861(88)90246-9.
Prostaglandin H synthase has two distinct catalytic activities: a cyclooxygenase activity that forms prostaglandin G2 from arachidonic acid; and a peroxidase activity that reduces prostaglandin G2 to prostaglandin H2. Lipid hydroperoxides, such as prostaglandin G2, also initiate the cyclooxygenase reaction, probably via peroxidase reaction cycle enzyme intermediates. The relation between the binding sites for lipid substrates of the two activities was investigated with an analysis of the effects of arachidonic and docosahexaenoic acids on the reaction kinetics of the peroxidase activity, and their effects on the ability of a lipid hydroperoxide to initiate the cyclooxygenase reaction. The cyclooxygenase activity of pure ovine synthase was found to have an apparent Km value for arachidonate of 5.3 microM and a Ki value (competetive inhibitor) for docosahexaenoate of 5.2 microM. When present at 20 microM neither fatty acid had a significant effect on the apparent Km value of the peroxidase for 15-hydroperoxyeicosatetraenoic acid: the values were 7.6 microM in the absence of docosahexaenoic acid and 5.9 microM in its presence, and (using aspirin-treated synthase) 13.7 microM in the absence of arachidonic acid and 15.7 microM in its presence. Over a range of 1 to 110 microM the level of arachidonate had no significant effect on the initiation of the cyclooxygenase reaction by 15-hydroperoxyeicosatetraenoic acid. The inability of either arachidonic acid or docosahexaenoic acid to interfere with the interaction between the peroxidase and lipid hydroperoxides indicates that the cyclooxygenase and peroxidase activities of prostaglandin H synthase have distinct binding sites for their lipid substrates.
前列腺素H合酶具有两种不同的催化活性：一种是环氧化酶活性，可将花生四烯酸转化为前列腺素G2；另一种是过氧化物酶活性，可将前列腺素G2还原为前列腺素H2。脂质氢过氧化物，如前列腺素G2，也可能通过过氧化物酶反应循环酶中间体引发环氧化酶反应。通过分析花生四烯酸和二十二碳六烯酸对过氧化物酶活性反应动力学的影响，以及它们对脂质氢过氧化物引发环氧化酶反应能力的影响，研究了这两种活性的脂质底物结合位点之间的关系。发现纯绵羊合酶的环氧化酶活性对花生四烯酸的表观Km值为5.3μM，对二十二碳六烯酸的Ki值（竞争性抑制剂）为5.2μM。当两种脂肪酸均以20μM存在时，对过氧化物酶对15-氢过氧化二十碳四烯酸的表观Km值均无显著影响：在不存在二十二碳六烯酸时该值为7.6μM，存在时为5.9μM，（使用阿司匹林处理的合酶）在不存在花生四烯酸时为13.7μM，存在时为15.7μM。在1至110μM范围内，花生四烯酸水平对15-氢过氧化二十碳四烯酸引发环氧化酶反应无显著影响。花生四烯酸或二十二碳六烯酸均无法干扰过氧化物酶与脂质氢过氧化物之间的相互作用，这表明前列腺素H合酶的环氧化酶和过氧化物酶活性对其脂质底物具有不同的结合位点。
