Pollegioni L, Porrini D, Molla G, Pilone M S
Department of Structural and Functional Biology, University of Insubria, Varese, Italy.
Eur J Biochem. 2000 Nov;267(22):6624-32. doi: 10.1046/j.1432-1327.2000.01757.x.
The redox potentials and pH characteristics of D-amino-acid oxidase (EC 1.4.3.3; DAAO) from the yeast Rhodotorula gracilis and Trigonopsis variabilis were measured in the pH range 6.5-8.5 at 15 degrees C. In the free enzyme form, the anionic red semiquinone is quantitatively formed in both DAAOs, indicating that a two single-electron transfer mechanism is active. The semiquinone species is also thermodynamically stable, as indicated by the large separation of the single-electron transfer potentials. The first electron potential is pH-independent, while the second electron transfer is pH-dependent exhibiting a approximately -60 mV/pH unit slope, consistent with a one-electron/one-proton transfer. In the presence of the substrate analogue benzoate, the two-electron transfer is the thermodynamically favoured process for both DAAOs, with only a quantitative difference in the stabilization of the anionic semiquinone. Clearly binding of the substrate (or substrate analogue) modulates the redox properties of the two enzymes. In both cases, in the presence and absence of benzoate, the slope of Em vs. pH (-30 mV/pH unit) corresponds to an overall two-electron/one-proton transfer in the reduction to yield the anionic reduced flavin. This behaviour is similar to that reported for DAAO from pig kidney. The differences in potentials and the stability of the semiquinone intermediate measured for the three DAAOs probably stem from different isoalloxazine environments. In the case of R. gracilis DAAO, the low stability of the semiquinone form in the DAAO-benzoate complex can be explained by the shift in position of the side chain of Arg285 following substrate analogue binding.
在15℃下，于pH 6.5 - 8.5范围内测定了来自纤细红酵母和可变三角酵母的D-氨基酸氧化酶（EC 1.4.3.3；DAAO）的氧化还原电位和pH特性。在游离酶形式下，两种DAAO中均定量形成阴离子红色半醌，表明双单电子转移机制是活跃的。半醌物种在热力学上也是稳定的，这由单电子转移电位的较大分离表明。第一个电子电位与pH无关，而第二个电子转移与pH有关，呈现出约-60 mV/pH单位的斜率，这与单电子/单质子转移一致。在底物类似物苯甲酸盐存在下，双电子转移是两种DAAO热力学上更有利的过程，只是在阴离子半醌的稳定性上存在定量差异。显然，底物（或底物类似物）的结合调节了这两种酶的氧化还原性质。在两种情况下，无论有无苯甲酸盐，Em对pH的斜率（-30 mV/pH单位）对应于还原生成阴离子还原黄素时的整体双电子/单质子转移。这种行为与猪肾DAAO报道的行为相似。三种DAAO测得的电位差异和半醌中间体的稳定性可能源于不同的异咯嗪环境。就纤细红酵母DAAO而言，DAAO - 苯甲酸盐复合物中半醌形式的低稳定性可以通过底物类似物结合后Arg285侧链位置的移动来解释。
