Van den Berghe-Snorek S, Stankovich M T
J Biol Chem. 1985 Mar 25;260(6):3373-9.
The redox properties of D-amino acid oxidase (D-amino-acid: O2 oxidoreductase (deaminating) EC1.4.3.3) have been measured at 18 degrees C in 20 mM sodium pyrophosphate, pH 8.5, and in 50 mM sodium phosphate, pH 7.0. Over the entire pH range, 2 eq are required per mol of FAD in D-amino acid oxidase for reduction to the anion dihydroquinone. The red anion semiquinone is thermodynamically stable as indicated by the separation of the electron potentials and the quantitative formation of the semiquinone species. The first electron potential is pH-independent at -0.098 +/- 0.004 V versus SHE while the second electron potential is pH-dependent exhibiting a 0.060 mV/pH unit slope. The redox behavior of D-amino acid oxidase is consistent with that observed for other oxidase enzymes. On the other hand, the behavior of the benzoate-bound enzyme under the same conditions is in marked contrast to the thermodynamics of free D-amino acid oxidase. Spectroelectrochemical experiments performed on inhibitor-bound (benzoate) D-amino acid oxidase show that benzoate binding regulates the redox properties of the enzyme, causing the energy levels of the benzoate-bound enzyme to be consistent with the two-electron transfer catalytic function of the enzyme. Our data are consistent with benzoate binding at the enzyme active site destroying the inductive effect of the positively charged arginine residue. Others have postulated that this positively charged group near the N(1)C(2) = O position of the flavin controls the enzyme properties. The data presented here are the clearest examples yet of enzyme regulation by substrate which may be a general characteristic of all flavoprotein oxidases.
已在18℃下，于pH 8.5的20 mM焦磷酸钠溶液以及pH 7.0的50 mM磷酸钠溶液中测定了D-氨基酸氧化酶（D-氨基酸：O2氧化还原酶（脱氨基），EC1.4.3.3）的氧化还原特性。在整个pH范围内，D-氨基酸氧化酶中每摩尔FAD还原为阴离子二氢醌需要2当量。如电子电位的分离和半醌物种的定量形成所示，红色阴离子半醌在热力学上是稳定的。第一个电子电位相对于标准氢电极（SHE）在-0.098±0.004 V时与pH无关，而第二个电子电位与pH有关，斜率为0.060 mV/pH单位。D-氨基酸氧化酶的氧化还原行为与其他氧化酶所观察到的一致。另一方面，相同条件下苯甲酸结合酶的行为与游离D-氨基酸氧化酶的热力学形成显著对比。对抑制剂结合（苯甲酸）的D-氨基酸氧化酶进行的光谱电化学实验表明，苯甲酸结合调节了酶的氧化还原特性，使苯甲酸结合酶的能级与酶的双电子转移催化功能一致。我们的数据与苯甲酸在酶活性位点的结合破坏带正电荷的精氨酸残基的诱导效应一致。其他人推测，黄素N(1)C(2)=O位置附近的这个带正电荷的基团控制着酶的性质。此处呈现的数据是底物对酶调控的最清晰实例之一，这可能是所有黄素蛋白氧化酶的一个普遍特征。
