Unraveling the Molecular Details of the Complete Mechanism That Governs the Synthesis of Prostaglandin G2 Catalyzed by Cyclooxygenase-2

Abstract

Cyclooxygenase-2 (COX-2) is the key enzyme involved in the synthesis pathway of prostaglandin G2 (PGG2) by transformation of arachidonic acid (AA). Although COX-2 is one of the principal pharmacological targets by the implication of PGG2 in several human diseases, the classical all-radical mechanism proposed for COX-2 catalysis has never been validated at the molecular level. Herein, molecular dynamics simulations and quantum mechanics/molecular mechanics (QM/MM) calculations were combined to analyze the six steps of the all-radical mechanism. The results show that O2 addition on C11 of AA can follow an antarafacial or suprafacial approach with respect to tyrosine 385, but only the antarafacial addition leads to the product with the correct 11R stereochemistry as established in the mechanistic proposal. Moreover, only the reaction pathway coming from the antarafacial intermediate describes a viable 8,12-cyclization to form the prostaglandin-like bicyclo endoperoxide that finally leads, by kinetic control, to PGG2 with the 15S stereochemistry found experimentally. The formation of the more stable trans ring isomer of natural PGG2 in an enzymatic environment is also explained. Our molecular analysis shows how COX-2 uses its relatively narrow channel in the active site to restrain certain conformational changes of AA and of the reaction intermediates, so that the PGG2 enzymatic synthesis turns out to be highly regiospecific and stereospecific. A more recent 10-step carbocation-based mechanistic proposal has been discarded.

The Biosynthesis of Enzymatically Oxidized Lipids

“…A second cyclization involving C8 and C12 generates a bicyclic ring and a radical at C15. Note that the cyclopentane ring is formed in the trans configuration (131). This differs from the non-enzymatically generated isoprostanes which occur predominantly in the cis configuration (132).…”

Unraveling how the Gly526Ser mutation arrests prostaglandin formation from arachidonic acid catalyzed by cyclooxygenase-2: a combined molecular dynamics and QM/MM study

“…This scenario is the opposite to the case of wild-type COX-2, where the suprafacial and antarafacial oxygen attacks produce the S and R congurations at C 11 , respectively. 13 On the other hand, the potential energy barriers (7.1 to 11.2 kcal mol À1 ) turn out to be small in comparison with that for hydrogen abstraction (24.8 kcal mol À1 ).…”

“…Our calculations conrm the experimental nding by Brash and co-workers, 16 who found that the 8,12-cyclization does not occur in the Gly526Ser mutant COX-2. We know that the cyclopentane ring closure only occurs with a (9R, 11R) conguration, 13,16 but this is just the stereochemical conguration that cannot be reached aer the third mechanistic step in the mutant, as mentioned above. The reason for that can be understood by analyzing Fig.…”

“…In a previous paper we studied 13 the mechanism that governs the biosynthesis of PGG 2 catalyzed by wild-type COX-2. That system was built from the murine COX-2/AA complex (PDB code 3HS5) 3 , replacing the Co 3+ complexed to the protoporphyrin IX group by Fe 3+ .…”

“…A comparison between the structure corresponding to snapshot 1 extracted from the MD simulation of the COX-2/AA Michaelis complex (see ref. 13) and the centroid snapshot of the most populated cluster of the last MD simulation of the Gly526Ser COX-2/AA Michaelis complex (Fig. 4) is pictured in Fig.…”