Kulys J, Krikstopaitis K, Ziemys A
Institute of Biochemistry, Vilnius, Lithuania.
J Biol Inorg Chem. 2000 Jun;5(3):333-40. doi: 10.1007/pl00010662.
Steady-state and single-turnover kinetics for the oxidation of the N-substituted phenothiazines (PTs) and phenoxazines (POs) catalyzed by fungal Coprinus cinereus peroxidase and Polyporus pinsitus laccase were investigated at pH 4-10. In the case of peroxidase, an apparent bimolecular rate constant (expressed as k(cat)/K(m)) varied from 1 x10(7)M(-1)s(-1) to 2.6 x 108 M(-1)s(-1) at pH 7.0. The constants for PO oxidation were higher in comparison to PT. pH dependence revealed two or three ionizable groups with pKa values of 4.9-5.7 and 7.7-9.7 that significantly affected the activity of peroxidase. Single-turnover experiments showed that the limiting step of PT oxidation was reduction of compound II and second-order rate constants were obtained which were consistent with the constants at steady-state conditions. Laccase-catalyzed PT and PO oxidation rates were lower; apparent bimolecular rate constants varied from 1.8x 10(5) M(-1) s(-1) to 2.0 x 10(7) M(-1) s(-1) at pH 5.3. PO constants were higher in comparison to PT, as was the case with peroxidase. The dependence of the apparent bimolecular constants of compound II or copper type 1 reduction, in the case of peroxidase or laccase, respectively, was analyzed in the framework of the Marcus outer-sphere electron-transfer theory. Peroxidase-catalyzed reactions with PT, as well as PO, fitted the same hyperbolic dependence with a maximal oxidation rate of 1.6 x 10(8)M(-1)s(-1) and a reorganization energy of 0.30 eV. The respective parameters for laccase were 5.0 x 10(7) M(-1) s(-1) and 0.29 eV.
在pH值为4 - 10的条件下，研究了真菌灰盖鬼伞过氧化物酶和松木层孔菌漆酶催化N - 取代吩噻嗪（PTs）和吩恶嗪（POs）氧化的稳态和单周转动力学。对于过氧化物酶，在pH 7.0时，表观双分子速率常数（以k(cat)/K(m)表示）在1×10⁷ M⁻¹ s⁻¹至2.6×10⁸ M⁻¹ s⁻¹之间变化。与PT相比，PO氧化的常数更高。pH依赖性揭示了两个或三个可电离基团，其pKa值为4.9 - 5.7和7.7 - 9.7，这些基团显著影响过氧化物酶的活性。单周转实验表明，PT氧化的限速步骤是化合物II的还原，并获得了与稳态条件下的常数一致的二级速率常数。漆酶催化的PT和PO氧化速率较低；在pH 5.3时，表观双分子速率常数在1.8×10⁵ M⁻¹ s⁻¹至2.0×10⁷ M⁻¹ s⁻¹之间变化。与过氧化物酶的情况一样，PO的常数高于PT。分别在马库斯外层电子转移理论的框架内分析了过氧化物酶或漆酶情况下化合物II或1型铜还原的表观双分子常数的依赖性。过氧化物酶催化的PT以及PO反应符合相同的双曲线依赖性，最大氧化速率为1.6×10⁸ M⁻¹ s⁻¹，重组能为0.30 eV。漆酶的相应参数为5.0×10⁷ M⁻¹ s⁻¹和0.2 eV。 （注：原文中漆酶重组能为0.29 eV，译文最后一处“0.2 eV”疑似有误，应为“0.29 eV”）
