Involvement of arginine 120, glutamate 524, and tyrosine 355 in the binding of arachidonate and 2-phenylpropionic acid inhibitors to the cyclooxygenase active site of ovine prostaglandin endoperoxide H synthase-1

Examination of the crystal structure of the ovine prostaglandin endoperoxide synthase-1 (PGHS-1)/S-flurbiprofen complex (Picot, D., Loll, P. J., and Garavito, R. M. (1994) Nature 367, 243-2491) suggests (a) that the carboxyl group of arachidonic acid interacts with the arginino group of Arg(120); (b) that Arg(120) forms an important salt bridge with Glu(524); and (c) that Tyr(355), which is in close proximity to Arg(120), could determine the stereochemical specificity of PGHS-1 toward 2-phenylpropionic acid inhibitors. To test these concepts, we used site-directed mutagenesis to prepare ovine PGHS-1 mutants having modifications of Arg(120) (R120K, R120Q, R120E), Glu(524) (E524D, E524Q, E524K), and Tyr(355) (Y355F) and examined the properties of the mutant enzymes expressed in COS-1 cells. All of the mutants retained at least part of their cyclooxygenase and peroxidase activities except the R120E mutant, which had no detectable activity. The K-m values of the R120K and R120Q mutants with arachidonic acid were 87 and 3300 mu M, respectively, versus 4 mu M for native PGHS-1. The R120Q mutant failed to undergo suicide inactivation during catalysis or time dependent inhibition by flurbiprofen. These results are consistent with Arg(120) binding the carboxylate group of arachidonate and suggest that interaction of the carboxylate group of substrates and inhibitors with Arg(120) is necessary for suicide inactivation and time-dependent inhibition, respectively. The K-m values for the E524D, E524Q, and E524K mutants were not significantly different from values obtained for the native PGHS-1, suggesting that this residue is not importantly involved in catalysis or substrate binding. The effect of modifications of Arg(120) and Tyr(355) on the stereospecificity of inhibitor binding were determined. Ratios of IC50 values for cyclooxygenase inhibition by D- and L-ibuprofen, a competitive cyclooxygenase inhibitor, were 32, 67, and 7.1 for native PGHS-1, R120Q PGHS-1, and Y355F PGHS-1, respectively. The decreased stereochemical specificity observed with the Y355F PGHS-1 mutant suggests that Tyr(355) is a determinant of the stereospecificity of PGHS-1 toward inhibitors of the 2-phenylpropionic acid class.