Human prostaglandin-endoperoxide H synthase-1 and -2 (hPGHS-1 and hPGHS-2) were expressed by transient transfection of COS-1 cells. Microsomes prepared from the transfected cells were used to measure the rates of oxygenation of several 18- and 20-carbon polyunsaturated fatty acid substrates including eicosapentaenoic, arachidonic, dihomo--linolenic, -linolenic (), -linolenic, and linoleic acids. Comparisons of k/Kvalues indicate that the order of efficiency of oxygenation is arachidonate > dihomo--linolenate > linoleate > -linolenate for both isozymes; while the order of efficiency was the same for hPGHS-1 and hPGHS-2, -linolenate was a particularly poor substrate for hPGHS-1. -Linolenate and eicosapentaenoate were poor substrates for both isozymes, but in each case, these two fatty acids were better substrates for hPGHS-2 than hPGHS-1. These studies of substrate specificities are consistent with previous studies of the interactions of PGHS isozymes with nonsteroidal anti-inflammatory drugs that have indicated that the cyclooxygenase active site of PGHS-2 is somewhat larger and more accommodating than that of PGHS-1. The major products formed from linoleate and -linolenate were characterized. 13-Hydroxy-(9Z,11E)-octadecadienoic acid was found to be the main product formed from -linoleate by both isozymes. The major products of oxygenation of -linolenate were determined by mass spectrometry to be 12-hydroxy-(9Z,13E/Z,15Z)-octadecatrienoic acids. This result suggests that -linolenate is positioned in the cyclooxygenase active site with a kink in the carbon chain such that hydrogen abstraction occurs from the 5-position in contrast to abstraction of the 8-hydrogen from other substrates.

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