Mutational and X-ray crystallographic analysis of the interaction of dihomo-gamma-linolenic acid with prostaglandin endoperoxide H syntheses

Prostaglandin endoperoxide H synthases-1 and -2 (PGHSs) catalyze the commit ted step in prostaglandin biosynthesis, Both isozymes can oxygenate a varie ty of related polyunsaturated fatty acids. We report here the x-ray crystal structure of dihomo-gamma -linolenic acid (DHLA) in the cyclooxygenase sit e of PGHS-1 and the effects of active site substitutions on the oxygenation of DHLA, and we compare these results to those obtained previously with ar achidonic acid (AA), DHLA is bound within the cyclooxygenase site in the sa me overall L-shaped conformation as Ak C-1 and C-11 through C-20 are in the same positions for both substrates, but the positions of C-2 through C-10 differ by up to 1.74 Angstrom In general, substitutions of active site resi dues caused parallel changes in the oxygenation of both AA and DHLA. Two si gnificant exceptions were Val-349 and Ser-530, A V349A substitution caused an 800-fold decrease in the V-max/K-m for DHLA but less than a a-fold chang e with AA; kinetic evidence indicates that C-13 of DHLA is improperly posit ioned with respect to Tyr-385 in the V349A mutant thereby preventing effici ent hydrogen abstraction. Val-349 contacts C-5 of DHLA and appears to serve as a structural bumper positioning the carboxyl half of DHLA, which, in tu rn, positions properly the omega -half of this substrate, A V349A substitut ion in PGHS-2 has similar, minor effects on the rates of oxygenation of AA and DHLA. Thus, Val-349 is a major determinant of substrate specificity for PGHS-1 but not for PGHS-2. Ser-530 also influences the substrate specifici ty of PGHS-1; an S530T substitution causes 40- and 760-fold decreases in ox ygenation efficiencies for AA and DHLA respectively.