Prostaglandin H synthase: Resolved and unresolved mechanistic issues

Abstract

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 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 Habstraction 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.Prostaglandin H synthase (PGHS) is a key enzyme in prostanoid biosynthesis. Mammalian systems have two distinct PGHS isozymes sharing ~60% sequence identity. The constitutive isozyme, PGHS-1, is thought to function usually as a housekeeping enzyme, whereas the inducible isozyme, PGHS-2, is associated with cytokine and mitogen dependent processes, such as inflammation and cell proliferation [1,2]. Both PGHS isozymes catalyze the same two reactions: dioxygenation of arachidonic acid (AA) to yield prostaglandin G 2 (PGG 2 ), containing both a 9-11 endoperoxide and a 15-peroxide group; and a peroxidase reaction, which converts PGG 2 to prostaglandin H 2 (PGH 2 ) where the 15-peroxide is reduced to an alcohol © 2009 Elsevier Inc. All rights reserved. *CORRESPONDING AUTHOR: FAX: 713 500-6810; Ah-Lim.Tsai@uth.tmc.edu.. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this e...

Enzyme Reactivation by Hydrogen Peroxide in Heme-based Tryptophan Dioxygenase

“…There are at least four tyrosine and tryptophan residues in the immediate vicinity of the enzyme active site. The identification of the radical site is challenging because a mutation of a tyrosine/tryptophan (fated to be a free radical) can result in the radical moving to a nearby tyrosine/tryptophan, as demonstrated in other heme-based enzymes such as prostaglandin H synthase (61).…”

Pre-existent Asymmetry in the Human Cyclooxygenase-2 Sequence Homodimer

“…The goal was to analyze enzyme forms in which the E allo and E cat monomers were clearly defined. A Tyr-385 radical is required to abstract a hydrogen atom from fatty acyl substrates in the first chemical step of COX catalysis (3,4). In principle, a Y385F/Native huPGHS-2 heterodimer can assume either an (E allo -Y385F/ E cat -Native) or an (E cat -Y385F/E allo -Native) form, but only the (E allo -Y385F/E cat -Native) version would, upon binding heme, be capable of COX catalysis (29).…”

“…Various amounts of guanidine hydrochloride were then added, and the samples were incubated at room temperature for 30 min. After centrifugation, the denaturant-treated proteins were incubated again with 1 mM [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]acetylsalicylate for 30 min at room temperature. Total aspirin acetylation was quantified by liquid scintillation counting as described above.…”

“…The structure/function relationships of PGHSs have been studied in considerable detail (1)(2)(3)(4). PGHSs are sequence homodimers.…”

Unique coupling of mono- and dioxygenase chemistries in a single active site promotes heme degradation

“…Dioxygenase active sites are generally hydrophobic as observed for tryptophan and indoleamine 2,3-dioxygenases, where O 2 binds ferrous heme iron akin to cytochrome P450, but perform distinct chemistry (3)(4)(5). Prostaglandin-endoperoxide synthase, also known as cyclooxygenase (COX), is another type of dioxygenase where a nonactivated O 2 molecule binds to a fatty acid radical generated by initial hydrogen abstraction (6,7). Whereas COX as well as linoleate diol synthase further convert the dioxygenated product with peroxidase or isomerase activity, respectively (6,8), each dioxygenase has another active site designed for the latter reaction.…”