DYNAMICS OF PROSTAGLANDIN-H SYNTHASES - STUDIES WITH PROSTAGLANDIN-H SYNTHASE-2 Y355F UNMASK MECHANISMS OF TIME-DEPENDENT INHIBITION AND ALLOSTERIC ACTIVATION
Oy. So et al., DYNAMICS OF PROSTAGLANDIN-H SYNTHASES - STUDIES WITH PROSTAGLANDIN-H SYNTHASE-2 Y355F UNMASK MECHANISMS OF TIME-DEPENDENT INHIBITION AND ALLOSTERIC ACTIVATION, The Journal of biological chemistry, 273(10), 1998, pp. 5801-5807
Prostaglandin H synthases (PGHSs) catalyze the conversion of arachidon
ic acid to prostaglandins. In this report, we describe the effect of a
PGHS2 Y355F mutation on the dynamics of PGHS2 catalysis and inhibitio
n, Tyr(355) is part of a hydrogen-bonding network located at the entra
nce to the cyclooxygenase active site, The Y355F mutant exhibited allo
steric activation kinetics in the presence of arachidonic acid that wa
s defined by a curved Eadie-Scatchard plot and a Hill coefficient of 1
.36 +/- 0.05, Arachidonic acid induced allosteric activation has not b
een directly observed with wild type PGHS2. The mutation also decrease
d the observed time-dependent inhibition by indomethacin, flurbiprofen
, RS-57067, and SC-57666, Detailed kinetic analysis showed that the Y3
55F mutation decreased the transition state energy associated with slo
w-binding inhibition (EI double dagger) relative to the energy associa
ted with catalysis (ES double dagger) by 1.33, 0.67, and 1.06 kcal/mol
, respectively, for indomethacin, flurbiprofen, and RS-57067, These ob
servations 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 hypot
hesize that the dynamic events are the equilibrium between relaxed and
tightened organizations of the hydrogen-bonding network at the entran
ce to the cyclooxygenase active site, It is these rearrangements that
control the rate of substrate binding and ultimately the rate of prost
aglandin formation.