Bakovic M, Dunford H B
Department of Chemistry, University of Alberta, Edmonton, Canada.
Biochem Cell Biol. 1996;74(1):117-24. doi: 10.1139/o96-013.
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.
以过氧化氢为底物，研究了前列腺素内过氧化物合酶的初级氧化产物化合物I的形成与pH值和温度的关系。结果分析表明，化合物I的形成受一个pKa为4.06±0.04的可电离基团影响。在3.5至9.1的pH范围内，过氧化氢的质子化形式比酶的非质子化形式更易发生反应，这表明酸碱催化对化合物I的形成很重要。在离子强度为0.1 M、温度为4.0±0.2℃的pH无关区域，酶与过氧化氢反应的二级速率常数为(4.6±0.2)×10⁵ M⁻¹ s⁻¹。在pH无关区域（pH 6.98）、离子强度恒定为0.1 M的条件下，研究了3.4至24.1℃温度对化合物I形成速率的影响。从温度依赖性获得的动力学参数如下：阿仑尼乌斯活化能，Ea = 102±5 kJ/mol；活化自由能，ΔG‡ = 36±3 kJ/mol；活化焓，ΔH‡ = 100±5 kJ/mol；活化熵，ΔS‡ = 215±9 J/mol K。这些活化值与其他过氧化物酶和过氧化氢酶与过氧化氢反应的活化值有很大不同，表明活性位点结构存在显著差异。
