FATTY-ACID SUBSTRATE SPECIFICITIES OF HUMAN PROSTAGLANDIN ENDOPEROXIDE-H SYNTHASE-1 AND SYNTHASE-2 - FORMATION OF 12-HYDROXY-(9Z,13E Z,15Z)-OCTADECATRIENOIC ACIDS FROM ALPHA-LINOLENIC ACID/

Human prostaglandin-endoperoxide H synthase-l and -2 (hPGHS-1 and hPGH S-2) were expressed by transient transfection of COS-1 cells. Microsom es 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-gamma-lino lenic, alpha-linolenic (Delta(9,12,15)), gamma-linolenic, and linoleic acids. Comparisons of k(cat)/K-m values indicate that the order of ef ficiency of oxygenation is arachidonate > dihomo-gamma-linolenate > li noleate > alpha-linolenate for both isozymes; while the order of effic iency was the same for hPGHS-1 and hPGHS-2, alpha-linoleate was a part icularly poor substrate for hPGHS-1. gamma-Linolenate and eicosapentae noate 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 studi es of the interactions of PGHS isozymes with nonsteroidal anti-inflamm atory 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 alpha-linolenate were ch aracterized. 13-Hydroxy-(9Z,11E)-octadecadienoic acid was found to be the main product formed from alpha-linoleate by both isozymes. The maj or products of oxygenation of alpha-linolenate were determined by mass spectrometry to be 12-hydroxy-(9Z,13E/Z,15Z)-octadecatrieno acids. Th is result suggests that alpha-linolenate is positioned in the cyclooxy genase active site with a kink in the carbon chain such that hydrogen abstraction occurs from the omega 5-position in contrast to abstractio n of the omega 8-hydrogen from other substrates.