PH AND TEMPERATURE-DEPENDENCE OF THE RATE OF COMPOUND-I FORMATION FROM THE REACTION OF PROSTAGLANDIN ENDOPEROXIDE SYNTHASE WITH HYDROGEN-PEROXIDE

The formation of primary oxidized compound of prostaglandin endoperoxi de synthase, compound I, was studied as a function of pH and temperatu re using hydrogen peroxide as a substrate. Analysis of the results ind icates that compound I formation is influenced by an ionizable group w ith a pK(a) of 4.06 +/- 0.04. The protonated form of hydrogen peroxide preferentially reacts with the unprotonated form of the enzyme over t he pH range of 3.5 to 9.1, suggesting the importance of acid-base cata lysis for compound I formation. The second-order rate constant for the reaction of the enzyme with hydrogen peroxide in the pH-independent r egion is (4.6 +/- 0.2) x 10(5) M(-1) s(-1) at an ionic strength of 0.1 M and temperature of 4.0 +/- 0.2 degrees C. The effect of temperature on the rate of compound I formation was studied from 3.4 to 24.1 degr ees C in the pH-independent region (pH 6.98) and at a constant ionic s trength of 0.1 M. The kinetic parameters obtained from the temperature dependence are the following: Arrhenius activation energy, E(a) = 102 +/- 5 kJ/mol; free energy of activation, Delta G(double dagger), 36 /- 3 kJ/mol; enthalpy of activation, Delta H-double dagger, 100 +/- 5 kJ/mol; entropy of activation, Delta S-double dagger, 215 +/- 9 J/mol K. These activation values are very different from those obtained for the reactions of other peroxidases and catalases with hydrogen peroxid e, indicating profound differences in active site structure.