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 endoperoxide synthase, compound I, was studied as a function of pH and temperature using hydrogen peroxide as a substrate. Analysis of the results indicates that compound I formation is influenced by an ionizable group with a pKa of 4.06 +/- 0.04. The protonated form of hydrogen peroxide preferentially reacts with the unprotonated form of the enzyme over the pH range of 3.5 to 9.1, suggesting the importance of acid-base catalysis for compound I formation. The second-order rate constant for the reaction of the enzyme with hydrogen peroxide in the pH-independent region 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 degrees C in the pH-independent region (pH 6.98) and at a constant ionic strength of 0.1 M. The kinetic parameters obtained from the temperature dependence are the following: Arrhenius activation energy, Ea = 102 +/- 5 kJ/mol; free energy of activation, delta G++, 36 +/- 3 kJ/mol; enthalpy of activation, delta H++, 100 +/- 5 kJ/mol; entropy of activation, delta S++, 215 +/- 9 J/mol K. These activation values are very different from those obtained for the reactions of other peroxidases and catalases with hydrogen peroxide, indicating profound differences in active site structure.