Abstract
Prostaglandin endoperoxide H synthase-2 (PGHS-2), also known as cyclooxygenase-2 (COX-2), is a sequence homodimer. However, the enzyme exhibits half-site heme and inhibitor binding and functions as a conformational heterodimer having a catalytic subunit (Ecat) with heme bound and an allosteric subunit (Eallo) lacking heme. Some recombinant heterodimers composed of a COX-deficient mutant subunit and a native subunit (i.e. Mutant/Native PGHS-2) have COX activities similar to native PGHS-2. This suggests that the presence of heme plus substrate leads to the subunits becoming lodged in a semi-stable Eallo-mutant/Ecat-Native∼heme form during catalysis. We examined this concept using human PGHS-2 dimers composed of combinations of Y385F, R120Q, R120A, and S530A mutant or native subunits. With some heterodimers (e.g. Y385F/Native PGHS-2), heme binds with significantly higher affinity to the native subunit. This correlates with near native COX activity for the heterodimer. With other heterodimers (e.g. S530A/Native PGHS-2), heme binds with similar affinities to both subunits, and the COX activity approximates that expected for an enzyme in which each monomer contributes equally to the net COX activity. With or without heme, aspirin acetylates one-half of the subunits of the native PGHS-2 dimer, the Ecat subunits. Subunits having an S530A mutation are refractory to acetylation. Curiously, aspirin acetylates only one-quarter of the monomers of S530A/Native PGHS-2 with or without heme. This implies that there are comparable amounts of two noninterchangeable species of apoenzymes, Eallo-S530A/Ecat-Native and Eallo-Native/Ecat-S530A. These results suggest that native PGHS-2 assumes a reasonably stable, asymmetric Eallo/Ecat form during its folding and processing.
Highlights
Cyclooxygenase-2 (COX-2), a target of coxibs, aspirin, and related drugs, is a sequence homodimer that functions as a conformational heterodimer
This suggests that the presence of heme plus substrate leads to the subunits becoming lodged in a semistable Eallo-mutant/Ecat-Nativeϳheme form during catalysis. We examined this concept using human Prostaglandin endoperoxide H synthase-2 (PGHS-2) dimers composed of combinations of Y385F, R120Q, R120A, and S530A mutant or native subunits
In terms of Vmax values, we determined the Vmax for O2 consumption experimentally using an O2 electrode in all cases. This value was normalized in some instances to a Vmax for arachidonic acid (AA) consumption by correcting for differences in the ratios of products derived from PGG2 versus hydroperoxyeicosatetraenoic acids (HPETEs)
Summary
Cyclooxygenase-2 (COX-2), a target of coxibs, aspirin, and related drugs, is a sequence homodimer that functions as a conformational heterodimer. Some recombinant heterodimers composed of a COX-deficient mutant subunit and a native subunit (i.e. Mutant/Native PGHS-2) have COX activities similar to native PGHS-2 This suggests that the presence of heme plus substrate leads to the subunits becoming lodged in a semistable Eallo-mutant/Ecat-Nativeϳheme form during catalysis. PGHSs are sequence homodimers, they exhibit half-sites heme and inhibitor binding and function as conformational heterodimers composed of Eallo and Ecat partner monomers [17,18,19,20,21,22,23,24,25]. Studies of aspirin acetylation with one particular variant, S530A/Native huPGHS-2, led us to the conclusion that PGHSs assume a stable conformational heterodimeric form relatively early in their lifetimes, i.e. during their folding and processing
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