Rönnberg M, Ellfolk N, Dunford H B
Acta Chem Scand B. 1984;38(1):79-83. doi: 10.3891/acta.chem.scand.38b-0079.
The effect of pH on the stability and overall catalytic activity of half-reduced Pseudomonas cytochrome c peroxidase was studied over the pH range 3.5-8. The stability of the enzyme as deduced from 40 s incubation experiments is virtually unaffected by pH. However, there is a bell-shaped pH dependence for the overall catalytic reaction using H2O2 as oxidizing substrate and cytochrome c-551 as reducing substrate with maximum turnover rate of pH 6. The effects of pH on (1) rate of reduction of the totally ferric enzyme by reduced azurin over the pH range 3.5-8 and (2) the rate of compound I formation from the half-reduced enzyme and hydrogen peroxide over the pH range 4-8 were also investigated. The reduction reaction rate also appears bell-shaped with optimum rate at pH 5.6. The rate of compound I formation is virtually pH independent above pH 5 but drops dramatically as the pH is lowered from 5 to 4. The influence of an ionization with apparent pKa value of 4.4 is implicated in compound I formation. This enzyme acid group must be deprotonated for compound I formation to occur suggesting the importance of base catalysis.
在pH值3.5 - 8范围内研究了pH对半还原的假单胞菌细胞色素c过氧化物酶稳定性和整体催化活性的影响。从40秒孵育实验推断，酶的稳定性实际上不受pH影响。然而，以H2O2作为氧化底物和细胞色素c - 551作为还原底物时，整体催化反应呈现钟形pH依赖性，在pH 6时周转速率最高。还研究了pH对以下方面的影响：(1) 在pH值3.5 - 8范围内，还原型天青蛋白对半还原酶的还原速率；(2) 在pH值4 - 8范围内，半还原酶与过氧化氢形成化合物I的速率。还原反应速率也呈钟形，在pH 5.6时速率最佳。在pH高于5时，化合物I的形成速率实际上与pH无关，但当pH从5降至4时，速率急剧下降。化合物I的形成涉及一个表观pKa值为4.4的电离作用。该酶酸性基团必须去质子化才能形成化合物I，这表明碱催化的重要性。
