Thr268 in substrate binding and catalysis in P450BM-3.
Thr268 in substrate binding and catalysis in P450BM-3.
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DOI：
10.1006/abbi.1997.0400
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发表时间：
1998
影响因子：
3.9
通讯作者：
G. Truan;J. Peterson
中科院分区：
文献类型：
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作者：
G. Truan;J. Peterson
被称为“P450”的蛋白质基因超家族的成员催化单加氧反应，每个催化循环需要输入两个电子和一个氧分子。这些蛋白质广泛分布于从细菌到人类的生物体中。P450 BM-3是一种从巨大芽孢杆菌中分离的可溶性蛋白，是自给自足的，在同一多肽上含有P450和还原酶结构域。P450 BM-3催化各种脂肪酸在omega-1、omega-2和omega-3位置的羟基化，以及双键的环氧化。我们构建了活性位点突变体T268 A，并分析了其对花生四烯酸和棕榈酸氧化的影响。数据表明，突变改变了花生四烯酸和棕榈酸氧化的偶联（消耗的NADPH与形成的产物的比率）。我们还分析了异丙苯过氧化氢驱动的反应，并表明它们不受这种突变的影响。这些数据，以及脂肪酸结合的研究，支持的假设的作用的I-螺旋残基，T268，在保持脂肪酸底物在正确的位置生产羟基化在催化循环的这种酶。
Members of the gene superfamily of proteins called "P450" catalyze monooxygenation reactions that require an input of two electrons and a molecule of oxygen per catalytic cycle. These proteins are widely distributed among living organisms, from bacteria to human. P450BM-3, a soluble protein isolated from Bacillus megaterium, is self-sufficient, containing P450 and reductase domains on the same polypeptide. P450BM-3 catalyzes the hydroxylation of various fatty acids at omega-1, omega-2, and omega-3 positions, as well as epoxidations of double bonds. We have constructed the active-site mutant, T268A, and analyzed the effect on arachidonic acid and palmitic acid oxidation. Data indicate that the mutation changes the coupling (ratio of NADPH consumed versus product formed) for both arachidonic acid and palmitic acid oxidation. We have also analyzed cumene hydroperoxide-driven reactions and shown that they are unaffected by this mutation. These data, as well as fatty acid binding studies, support the hypothesis of a role of the I-helix residue, T268, in maintaining fatty acid substrates in the correct position for productive hydroxylation during the catalytic cycle of this enzyme.