Tsai A L, Wu G, Kulmacz R J
Division of Hematology, Department of Internal Medicine, University of Texas Health Science Center at Houston, 6431 Fannin Street, Houston, Texas 77030, USA.
Biochemistry. 1997 Oct 21;36(42):13085-94. doi: 10.1021/bi970397s.
Prostaglandin H synthase (PGHS) catalyzes both peroxidase and cyclooxygenase reactions. Resolution of several current issues regarding the PGHS catalytic mechanism hinges on the stoichiometry of the reaction of PGHS with hydroperoxide, fatty acid, and oxygen. The dependence of wide-doublet tyrosyl radical accumulation in PGHS isoform 1 on hydroperoxide stoichiometry, has been determined; this catalytically active radical is formed efficiently at stoichiometries </=1 after only 300 ms of reaction. This is consistent with intramolecular formation of the radical from PGHS Compound I but inconsistent with an alternative pathway involving reduction of Compound I to Compound II by a second hydroperoxide molecule. Results from stopped-flow studies indicate that the hydroperoxide level influences the rate of Compound II formation indirectly, via changes in the transient accumulation of Compound I, rather than by reducing Compound I. PGHS and soybean lipoxygenase reactions with 11,14-eicosadienoic acid (20:2) were also analyzed using a spectrophotometer cuvette fitted with an oxygen electrode to monitor lipid product formation and oxygen consumption simultaneously. The results show that the oxygen electrode signal is inherently dampened and thus underestimates the oxygen consumption rate; the discrepancy is much larger for the more rapidly accelerating PGHS reaction than for the lipoxygenase reaction. When correction is made for the electrode dampening, the ratio between the peak rates of oxygen consumption and lipid product formation was near unity for both PGHS and lipoxygenase, indicating a reaction stoichiometry of about 1 mol of O2 consumed/mol of 20:2 oxygenated for both enzymes. Separately, a stoichiometry of 0.9 mol of O2 consumed / mol oxygenated fatty acid was obtained when limiting amounts of 20:2 were reacted to completion with excess PGHS; the corresponding stoichiometry with arachidonic acid was 1.9. These O2/fatty acid stoichiometries are consistent with a dioxygenase mechanism for reaction of PGHS with both fatty acids and inconsistent with a mixed dioxygenase/monooxygenase mechanism proposed for the reaction with 20:2. The present conclusions reduce the complexity of the mechanisms that need to be considered for PGHS catalysis.
前列腺素H合酶（PGHS）催化过氧化物酶和环氧化酶反应。解决当前有关PGHS催化机制的几个问题取决于PGHS与氢过氧化物、脂肪酸和氧气反应的化学计量关系。已确定PGHS同工型1中宽双峰酪氨酸自由基积累对氢过氧化物化学计量关系的依赖性；仅在反应300毫秒后，当化学计量比≤1时，这种具有催化活性的自由基就能高效形成。这与PGHS化合物I分子内形成自由基一致，但与另一种通过第二个氢过氧化物分子将化合物I还原为化合物II的途径不一致。停流研究结果表明，氢过氧化物水平通过化合物I瞬态积累的变化间接影响化合物II的形成速率，而不是通过还原化合物I。还使用配备氧电极的分光光度计比色皿分析了PGHS和大豆脂氧合酶与11,14-二十碳二烯酸（20:2）的反应，以同时监测脂质产物形成和氧气消耗。结果表明，氧电极信号本身会被衰减，因此会低估氧气消耗速率；对于加速更快的PGHS反应，这种差异比脂氧合酶反应大得多。当对电极衰减进行校正后，PGHS和脂氧合酶的氧气消耗峰值速率与脂质产物形成峰值速率之比接近1，表明两种酶的反应化学计量关系约为每摩尔20:2氧化消耗1摩尔O2。另外，当有限量的20:2与过量的PGHS反应至完全时，得到的化学计量关系为每摩尔氧化脂肪酸消耗0.9摩尔O2；与花生四烯酸相应的化学计量关系为1.9。这些O2/脂肪酸化学计量关系与PGHS与两种脂肪酸反应的双加氧酶机制一致，与针对与20:2反应提出的混合双加氧酶/单加氧酶机制不一致。目前的结论降低了PGHS催化所需考虑机制的复杂性。
