Department of Internal Medicine, University of Texas Health Science Center at Houston, Houston, Texas 77030, USA.
Arch Biochem Biophys. 2010 Jan 1;493(1):103-24. doi: 10.1016/j.abb.2009.08.019. Epub 2009 Sep 1.
The cyclooxygenase and peroxidase activities of prostaglandin H synthase (PGHS)-1 and -2 have complex kinetics, with the cyclooxygenase exhibiting feedback activation by product peroxide and irreversible self-inactivation, and the peroxidase undergoing an independent self-inactivation process. The mechanistic bases for these complex, non-linear steady-state kinetics have been gradually elucidated by a combination of structure/function, spectroscopic and transient kinetic analyses. It is now apparent that most aspects of PGHS-1 and -2 catalysis can be accounted for by a branched chain radical mechanism involving a classic heme-based peroxidase cycle and a radical-based cyclooxygenase cycle. The two cycles are linked by the Tyr385 radical, which originates from an oxidized peroxidase intermediate and begins the cyclooxygenase cycle by abstracting a hydrogen atom from the fatty acid substrate. Peroxidase cycle intermediates have been well characterized, and peroxidase self-inactivation has been kinetically linked to a damaging side reaction involving the oxyferryl heme oxidant in an intermediate that also contains the Tyr385 radical. The cyclooxygenase cycle intermediates are poorly characterized, with the exception of the Tyr385 radical and the initial arachidonate radical, which has a pentadiene structure involving C11-C15 of the fatty acid. Oxygen isotope effect studies suggest that formation of the arachidonate radical is reversible, a conclusion consistent with electron paramagnetic resonance spectroscopic observations, radical trapping by NO, and thermodynamic calculations, although moderate isotope selectivity was found for the H-abstraction step as well. Reaction with peroxide also produces an alternate radical at Tyr504 that is linked to cyclooxygenase activation efficiency and may serve as a reservoir of oxidizing equivalent. The interconversions among radicals on Tyr385, on Tyr504, and on arachidonate, and their relationships to regulation and inactivation of the cyclooxygenase, are still under active investigation for both PGHS isozymes.
前列腺素 H 合酶（PGHS）-1 和 -2 的环氧化酶和过氧化物酶活性具有复杂的动力学，环氧化酶表现出产物过氧化物的反馈激活和不可逆的自我失活，而过氧化物酶则经历独立的自我失活过程。通过结构/功能、光谱和瞬态动力学分析的组合，逐渐阐明了这些复杂的非线性稳态动力学的机制基础。现在，PGHS-1 和 -2 催化的大多数方面都可以通过涉及经典血红素过氧化物酶循环和基于自由基的环氧化酶循环的分支链自由基机制来解释。这两个循环通过 Tyr385 自由基连接，该自由基源自氧化的过氧化物酶中间产物，并通过从脂肪酸底物中提取氢原子开始环氧化酶循环。过氧化物酶循环中间产物已得到很好的描述，而过氧化物酶的自我失活与涉及中间产物中氧铁血红素氧化剂的破坏性副反应在动力学上相关联，该中间产物还包含 Tyr385 自由基。除了 Tyr385 自由基和初始花生四烯酸自由基外，环氧化酶循环中间产物的特征较差，花生四烯酸自由基具有涉及脂肪酸的 C11-C15 的五烯结构。氧同位素效应研究表明，花生四烯酸自由基的形成是可逆的，这一结论与电子顺磁共振光谱观察、NO 的自由基捕获以及热力学计算一致，尽管 H 提取步骤也发现了中等的同位素选择性。与过氧化物的反应还会在 Tyr504 处产生与环氧化酶激活效率相关的另一个自由基，并且可能作为氧化当量的储库。Tyr385、Tyr504 和花生四烯酸上自由基的相互转化及其与环氧化酶调节和失活的关系，仍然是两种 PGHS 同工酶的活跃研究领域。
