Substrate Interactions in the Cyclooxygenase-1 Active Site

Abstract

Prostaglandin endoperoxide H synthases-1 and -2 (PGHS-1 and -2) catalyze the conversion of arachidonic acid, two molecules of O₂ and two electrons to PGH₂. This is the committed step in the formation of prostaglandins and thromboxane A₂ [1]. Crystallographic studies of enzyme inhibitor complexes have suggested that the cyclooxygenase active sites of PGHSs are hydrophobic channels that protrude into the body of the major globular domain of the enzymes [2]. We have now determined the structure of arachidonic acid (AA) bound within the cyclooxygenase active site of ovine (o)PGHS-1 [3]. AA is bound in an extended L-shaped conformation and makes a total of 49 hydrophobic contacts (i.e. 2.5–4.0 Å) and two hydrophilic contacts with the protein involving a total of 19 different residues (Figures 1,2). Although AA can assume some 10⁷ low energy conformations [4], only three of these are catalytically competent [5]. One conformation leads to PGG₂, one leads to 11R-HPETE, and a third leads to 15R- plus 15S-HPETE. Previous mutational studies have established the importance of Arg120 in AA binding to PGHS-1 and -2 [6–8], the role of Tyr385 in abstraction of the 13-proS-hydrogen from AA [9,10] and the importance of Ser530 and I1e523 as determinants of inhibitor specificity [11–13]. We have performed mutational analyses of a number of the residues that line the cyclooxygenase channel to determine their functional importance in AA binding and oxygenation. Substitutions of several cyclooxygenase site residues lead to large increases in 11-HPETE or 15-HPETE formation but with small changes in the K_m for AA. Our results suggest that individually and collectively the hydrophobic residues function primarily to position AA in a specific conformation that optimizes its conversion to PGG₂.