PRE-STEADY-STATE KINETICS AND MODELING OF THE OXYGENASE AND CYCLOOXYGENASE REACTIONS OF PROSTAGLANDIN ENDOPEROXIDE SYNTHASE

The pre-steady-state kinetics of the prostaglandin endoperoxide syntha se oxygenase reaction with eicosadienoic acids and the cyclooxygenase reaction with arachidonic acid were investigated by stopped-flow spect rophotometry at 426 nm, an isosbestic point between native enzyme and compound I. A similar reaction mechanism for both types of catalysis i s defined from combined kinetic experiments and numerical simulations. In the first step a fatty acid hydroperoxide reacts with the native e nzyme to form compound I and the fatty acid hydroxide. In the second s tep the fatty acid reduces compound I to compound II and a fatty acid carbon radical is formed. This is followed by two fast steps: (1) the addition of either one molecule of oxygen (the oxygenase reaction) or two molecules of oxygen (the cyclooxygenase reaction) to the fatty aci d carbon radical to form the corresponding hydroperoxyl radical, and ( 2) the reaction of the hydroperoxyl radical with compound II to form t he fatty acid hydroperoxide and a compound I-protein radical. A unimol ecular reaction of the compound I-protein radical to reform the native enzyme is assumed for the last step in the cycle. This is a slow reac tion not significantly affecting steps 1 and 2 under pre-steady-state conditions. A linear dependence of the observed pseudo-first-order rat e constant, k(obs), an fatty acid concentration is quantitatively repr oduced by the model for both the oxygenase and cyclooxygenase reaction s. The simulated second order rate constants for the conversion of nat ive enzyme to compound I with arachidonic or eicosadienoic acids hydro peroxides as a substrate are 8 X 10(7) and 4 x 10(7) M(-1) s(-1), resp ectively. The simulated and experimentally obtained second-order rate constants for the conversion of compound I to compound II with arachid onic and eicosadienoic acids as a substrate are 1.2 x 10(5) and 3.0 X 10(5) M(-1) s(-1), respectively.