Dynamics of prostaglandin H synthases - Studies with prostaglandin H synthase 2 Y355F unmask mechanisms of time-dependent inhibition and allosteric activation

Prostaglandin H synthases (PGHSs) catalyze the conversion of arachidonic acid to prostaglandins. In this report, we describe the effect of a PGHS2 Y355F mutation on the dynamics of PGHS2 catalysis and inhibition, Tyr(355) is part of a hydrogen-bonding network located at the entrance to the cyclooxygenase active site, The Y355F mutant exhibited allosteric activation kinetics in the presence of arachidonic acid that was defined by a curved Eadie-Scatchard plot and a Hill coefficient of 1.36 +/- 0.05, Arachidonic acid induced allosteric activation has not been directly observed with wild type PGHS2. The mutation also decreased the observed time-dependent inhibition by indomethacin, flurbiprofen, RS-57067, and SC-57666, Detailed kinetic analysis showed that the Y355F mutation decreased the transition state energy associated with slow-binding inhibition (EI double dagger) relative to the energy associated with catalysis (ES double dagger) by 1.33, 0.67, and 1.06 kcal/mol, respectively, for indomethacin, flurbiprofen, and RS-57067, These observations show Tyr(355) to be involved in the molecular mechanism of time-dependent inhibition, We interpret these results to indicate that slow binding inhibitors and the Y355F mutant slow the rate and unmask intrinsic, dynamic events associated with product formation, We hypothesize that the dynamic events are the equilibrium between relaxed and tightened organizations of the hydrogen-bonding network at the entrance to the cyclooxygenase active site, It is these rearrangements that control the rate of substrate binding and ultimately the rate of prostaglandin formation.