Kobayashi K, Miki M, Okamoto K, Nishino T
Institute of Scientific and Industrial Research, Osaka University, Japan.
J Biol Chem. 1993 Nov 25;268(33):24642-6.
The reduction of milk xanthine dehydrogenase by salicylate anion radical (SL-), nicotinamide adenine dinucleotide radical (NAD.), and 1-methylnicotinamide (NMA) radicals was investigated by the use of pulse radiolysis. Reduction of the dehydrogenase with SL- proceeded via two phases. From the kinetic difference spectra obtained, the faster and slower phases of reduction represent that of one of the iron-sulfur centers and of FAD, respectively. The rate constant of the faster phase increased with the concentration of the enzyme, suggesting that the reduction follows a bimolecular reaction of SL- with the iron-sulfur center. In contrast, the rate constant of the slower phase (510 s-1) was independent of the concentration of the enzyme at pH 7.5. In order to elucidate the contribution of the molybdenum site in the reaction, a similar reaction was performed with enzyme modified with oxipurinol. In the modified enzyme, the slower phase was lost, whereas the faster phase was not affected. These results suggest that the slower phase is due to intramolecular electron transfer from the molybdenum center to FAD. On the other hand, NAD. reacted predominantly with FAD of the dehydrogenase to form the neutral semiquinone of FAD with a second order rate constant of 1.4 x 10(7) M-1 s-1 at pH 7.5, whereas a similar reaction in the oxidase, which was converted from xanthine dehydrogenase by proteolytical cleavage, was not observed. This suggests that NAD. transfers an electron via the binding site for NAD+ on the dehydrogenase. In contrast, NMA radical reduced only an iron-sulfur center of the dehydrogenase with a second order rate constant of 6.5 x 10(7) M-1 s-1 at pH 7.5.
利用脉冲辐解研究了水杨酸根阴离子自由基（SL-）、烟酰胺腺嘌呤二核苷酸自由基（NAD·）和1-甲基烟酰胺（NMA）自由基对牛奶黄嘌呤脱氢酶的还原作用。SL-对脱氢酶的还原分两个阶段进行。从所得的动力学差异光谱来看，较快和较慢的还原阶段分别代表铁硫中心之一和黄素腺嘌呤二核苷酸（FAD）的还原阶段。较快阶段的速率常数随酶浓度增加而增大，这表明还原过程是SL-与铁硫中心的双分子反应。相比之下，在pH 7.5时，较慢阶段的速率常数（510 s-1）与酶浓度无关。为了阐明钼位点在该反应中的作用，用氧嘌呤醇修饰酶后进行了类似反应。在修饰后的酶中，较慢阶段消失，而较快阶段不受影响。这些结果表明，较慢阶段是由于分子内电子从钼中心转移到FAD所致。另一方面，NAD·主要与脱氢酶的FAD反应，在pH 7.5时形成FAD的中性半醌，二级速率常数为1.4×10⁷ M⁻¹ s⁻¹，而在通过蛋白水解裂解从黄嘌呤脱氢酶转化而来的氧化酶中未观察到类似反应。这表明NAD·通过脱氢酶上NAD⁺的结合位点转移电子。相比之下，NMA自由基仅以6.5×10⁷ M⁻¹ s⁻¹的二级速率常数在pH 7.5时还原脱氢酶的一个铁硫中心。
