Abstract
Prostaglandin endoperoxide H synthases (PGHSs) 1 and 2, also known as cyclooxygenases (COXs), catalyze the oxygenation of arachidonic acid (AA) in the committed step in prostaglandin (PG) biosynthesis. PGHSs are homodimers that display half of sites COX activity with AA; thus, PGHSs function as conformational heterodimers. Here we show that, during catalysis, fatty acids (FAs) are bound at both COX sites of a PGHS-2 dimer. Initially, an FA binds with high affinity to one COX site of an unoccupied homodimer. This monomer becomes an allosteric monomer, and it causes the partner monomer to become the catalytic monomer that oxygenates AA. A variety of FAs can bind with high affinity to the COX site of the monomer that becomes the allosteric monomer. Importantly, the efficiency of AA oxygenation is determined by the nature of the FA bound to the allosteric monomer. When tested with low concentrations of saturated and monounsaturated FAs (e.g. oleic acid), the rates of AA oxygenation are typically 1.5-2 times higher with PGHS-2 than with PGHS-1. These different kinetic behaviors of PGHSs may account for the ability of PGHS-2 but not PGHS-1 to efficiently oxygenate AA in intact cells when AA is a small fraction of the FA pool such as during "late phase" PG synthesis.
Highlights
POX site needs to be oxidized by a peroxide to oxidize Tyr-385 in the active COX site to a tyrosyl radical
We recently found in testing Prostaglandin endoperoxide H synthases (PGHSs)-2 with arachidonic acid (AA) and another COX substrate eicosapentaenoic acid (EPA) that the enzyme showed a marked preference for AA [8]
The average specific activities of preparations used in the experiments of purified ovPGHS-1 and huPGHS-2 with AA averaged 40 and 42 nmol of O2 consumed per min per mg of protein, respectively, under standard assay conditions
Summary
Materials—All fatty acids and their derivatives except 20:19 were purchased from Cayman Chemical Co. The methyl esters were saponified with KOH in ethanol-water as described previously for polyunsaturated fatty acids [9] and separated by preparative HPLC on a C18-reverse phase column (22 ϫ 250 mm, 5 m) with a mobile phase of acetonitrile-water-acetic acid (85:15:0.1, v/v) and a flow rate 30 ml/min to yield 98ϩ% of the target fatty acid as a colorless wax. The nickel-nitrilotriacetic acid elution fraction, which contained doubly His6-tagged homodimers and heterodimers that were both FLAG- and His6-tagged, was subjected to buffer-exchange with Buffer F (50 mM Tris-HCl, pH 7.5, containing 150 mM NaCl) and further purified using FLAG affinity resin following the protocol of the manufacturer. POX activity was measured spectrophotometrically using 0.1 mM H2O2 and 4 mM guaiacol as substrates [6]
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