Self W T, Stadtman T C
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Proc Natl Acad Sci U S A. 2000 Jun 20;97(13):7208-13. doi: 10.1073/pnas.97.13.7208.
During purification of the selenium-dependent xanthine dehydrogenase (XDH) from Clostridium purinolyticum, another hydroxylase was uncovered that also contained selenium and exhibited similar spectral properties. This enzyme was purified to homogeneity. It uses purine, 2OH-purine, and hypoxanthine as substrates, and based on its substrate specificity, this selenoenzyme is termed purine hydroxylase (PH). The product of hydroxylation of purine by PH is xanthine. A concomitant release of selenium from the enzyme and loss of catalytic activity on treatment with cyanide indicates that selenium is essential for PH activity. Selenium-dependent XDH, also purified from C. purinolyticum, was found to be insensitive to oxygen during purification and to use both potassium ferricyanide and 2,6-dichloroindophenol as electron acceptors. Selenium is required for the xanthine-dependent reduction of 2, 6-dichloroindophenol by XDH. Kinetic analyses of both enzymes revealed that xanthine is the preferred substrate for XDH and purine and hypoxanthine are preferred by PH. This characterization of these selenium-requiring hydroxylases involved in the interconversion of purines describes an extension of the pathway for purine fermentation in the purinolytic clostridia.
在从解嘌呤梭菌中纯化依赖硒的黄嘌呤脱氢酶（XDH）的过程中，发现了另一种羟化酶，它也含有硒并表现出相似的光谱特性。该酶被纯化至同质。它以嘌呤、2-羟基嘌呤和次黄嘌呤为底物，基于其底物特异性，这种含硒酶被称为嘌呤羟化酶（PH）。PH催化嘌呤羟化的产物是黄嘌呤。用氰化物处理时，酶中硒的伴随释放和催化活性的丧失表明硒对PH活性至关重要。同样从解嘌呤梭菌中纯化得到的依赖硒的XDH在纯化过程中对氧气不敏感，并以铁氰化钾和2,6-二氯靛酚作为电子受体。XDH依赖黄嘌呤还原2,6-二氯靛酚需要硒。两种酶的动力学分析表明，黄嘌呤是XDH的首选底物，而嘌呤和次黄嘌呤是PH的首选底物。对这些参与嘌呤相互转化的需硒羟化酶的表征描述了解嘌呤梭菌中嘌呤发酵途径的扩展。
