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

© 2019 American Chemical Society. 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.