Lu G, Tsai A L, Van Wart H E, Kulmacz R J
Department of Internal Medicine, University of Texas Health Science Center at Houston, Houston, Texas 77030, USA.
J Biol Chem. 1999 Jun 4;274(23):16162-7. doi: 10.1074/jbc.274.23.16162.
Prostaglandin H synthase isoforms 1 and 2 (PGHS-1 and -2) each have a peroxidase activity and also a cyclooxygenase activity that requires initiation by hydroperoxide. The hydroperoxide initiator requirement for PGHS-2 cyclooxygenase is about 10-fold lower than for PGHS-1 cyclooxygenase, and this difference may contribute to the distinct control of cellular prostanoid synthesis by the two isoforms. We compared the kinetics of the initial peroxidase steps in PGHS-1 and -2 to quantify mechanistic differences between the isoforms that might contribute to the difference in cyclooxygenase initiation efficiency. The kinetics of formation of Intermediate I (an Fe(IV) species with a porphyrin free radical) and Intermediate II (an Fe(IV) species with a tyrosyl free radical, thought to be the crucial oxidant in cyclooxygenase catalysis) were monitored at 4 degrees c by stopped flow spectrophotometry with several hydroperoxides as substrate. With 15-hydroperoxyeicosatetraenoic acid, the rate constant for Intermediate I formation (k1) was 2.3 x 10(7) M-1 s-1 for PGHS-1 and 2.5 x 10(7) M-1 s-1 for PGHS-2, indicating that the isoforms have similar initial reactivity with this lipid hydroperoxide. For PGHS-1, the rate of conversion of Intermediate I to Intermediate II (k2) became the limiting factor when the hydroperoxide level was increased, indicating a rate constant of 10(2)-10(3) s-1 for the generation of the active cyclooxygenase species. For PGHS-2, however, the transition between Intermediates I and II was not rate-limiting even at the highest hydroperoxide concentrations tested, indicating that the k2 value for PGHS-2 was much greater than that for PGHS-1. Computer modelling predicted that faster formation of the active cyclooxygenase species (Intermediate II) or increased stability of the active species increases the resistance of the cyclooxygenase to inhibition by the intracellular hydroperoxide scavenger, glutathione peroxidase. Kinetic differences between the PGHS isoforms in forming or stabilizing the active cyclooxygenase species can thus contribute to the difference in the regulation of their cellular activities.
前列腺素H合成酶同工型1和2（PGHS - 1和 - 2）各自具有过氧化物酶活性以及需要氢过氧化物引发的环氧化酶活性。PGHS - 2环氧化酶对氢过氧化物引发剂的需求比对PGHS - 1环氧化酶的需求低约10倍，这种差异可能导致这两种同工型对细胞前列腺素合成的不同调控。我们比较了PGHS - 1和 - 2中初始过氧化物酶步骤的动力学，以量化同工型之间可能导致环氧化酶引发效率差异的机制差异。在4℃下，通过停流分光光度法，以几种氢过氧化物为底物，监测中间体I（一种具有卟啉自由基的Fe(IV)物种）和中间体II（一种具有酪氨酸自由基的Fe(IV)物种，被认为是环氧化酶催化中的关键氧化剂）的形成动力学。对于15 - 氢过氧化二十碳四烯酸，PGHS - 1形成中间体I的速率常数（k1）为2.3×10⁷ M⁻¹ s⁻¹，PGHS - 2为2.5×10⁷ M⁻¹ s⁻¹，表明这两种同工型与这种脂质氢过氧化物具有相似的初始反应性。对于PGHS - 1，当氢过氧化物水平增加时，中间体I向中间体II的转化速率（k2）成为限制因素，表明生成活性环氧化酶物种的速率常数为10² - 10³ s⁻¹。然而，对于PGHS - 2，即使在测试的最高氢过氧化物浓度下，中间体I和II之间的转变也不是限速的，这表明PGHS - 2的k2值远大于PGHS - 1的k2值。计算机建模预测，活性环氧化酶物种（中间体II）的更快形成或活性物种稳定性的增加会增加环氧化酶对细胞内氢过氧化物清除剂谷胱甘肽过氧化物酶抑制的抗性。因此，PGHS同工型在形成或稳定活性环氧化酶物种方面的动力学差异可导致它们细胞活性调控的差异。
