Fenoll Lorena G, García-Molina Francisco, Gilabert María A, Varón Ramón, García-Ruiz Pedro A, Tudela José, García-Cánovas Francisco, Rodríguez-López José N
Grupo de Investigación Enzimología (GENZ), Departamento de Bioquímica y Biología Molecular-A, Facultad de Veterinaria, Universidad de Murcia, A. Correos 4021, E-30080 Murcia, Spain.
Biol Chem. 2005 Apr;386(4):351-60. doi: 10.1515/BC.2005.042.
The catalytic cycle of heme peroxidases involves three processes: the formation of compound I, its conversion to compound II and regeneration of the native enzyme. Each of the processes consists of a reversible binding stage followed by an irreversible transformation stage. Our group has proposed a continuous, sensitive and reliable chronometric method for measuring the steady-state rate of peroxidase activity. Furthermore, we have derived an analytical expression for the steady-state rate and simplified it, taking into consideration the experimental values of the rate constants of some stages previously determined by other authors in stopped-flow assays. We determined the value of the constant for the transformation of a series of phenols and anilines by compound II, and found that it involves a deprotonation step and an electron transfer step. Study of the solvent deuterium isotope effect on the oxidation of phenol revealed the non-rate-limiting character of the deprotonation step in a proton inventory study. Usage of the Marcus equation showed that the electronic transfer step is rate-limiting in both cases, while phenols and anilines were oxidised at different rates for the same potentials. This can be attributed to the shorter electron-tunnelling distance for electron transfer to the iron ion in the phenols than in the anilines.
化合物I的形成、其向化合物II的转化以及天然酶的再生。每个过程都由一个可逆结合阶段和一个不可逆转化阶段组成。我们小组提出了一种连续、灵敏且可靠的计时方法来测量过氧化物酶活性的稳态速率。此外，我们推导了稳态速率的解析表达式并进行了简化，同时考虑了其他作者先前在停流实验中测定的某些阶段速率常数的实验值。我们测定了化合物II对一系列酚类和苯胺类进行转化的常数，发现这涉及一个去质子化步骤和一个电子转移步骤。对酚类氧化过程中溶剂氘同位素效应的研究在质子累积研究中揭示了去质子化步骤的非限速特性。使用马库斯方程表明，在这两种情况下电子转移步骤都是限速步骤，而在相同电位下酚类和苯胺类的氧化速率不同。这可归因于酚类中电子转移到铁离子的电子隧穿距离比苯胺类中的短。
