MACROPHAGE NITRIC-OXIDE SYNTHASE - RELATIONSHIP BETWEEN ENZYME-BOUND TETRAHYDROBIOPTERIN AND SYNTHASE ACTIVITY
MACROPHAGE NITRIC-OXIDE SYNTHASE - RELATIONSHIP BETWEEN ENZYME-BOUND TETRAHYDROBIOPTERIN AND SYNTHASE ACTIVITY
复制标题
DOI：
10.1021/bi00146a019
复制
发表时间：
1992-08-11
期刊：
影响因子：
2.9
通讯作者：
MARLETTA, MA
中科院分区：
文献类型：
--
作者：
HEVEL, JM;MARLETTA, MA
一氧化氮合酶（NOS）（EC 1.14.23）催化L-精氨酸氧化为瓜氨酸和一氧化氮。由NOS进行的复杂反应涉及NADPH、O2、酶结合的FAD、FMN和四氢生物蝶呤（BH 4），直到最近才开始阐明。在这里，我们报告的小鼠巨噬细胞NOS的蝶呤需求的表征。虽然纯化的NOS活性不依赖于BH 4，活性显着增强BH 4浓度依赖性的方式。NOS纯化添加的BH 4的情况下，被发现含有亚化学计量浓度的酶结合的蝶呤，其中结合蝶呤的浓度增加与活性的增加，当在不存在外源性BH 4测定。然而，在BH 4的存在下，然后通过凝胶过滤纯化的NOS表现出1摩尔的蝶呤：1摩尔的NOS 130-kDa亚基的化学计量和活性，基本上是独立的外源性BH 4。使用蝶呤类似物进行实验以探测蝶呤的氧化还原作用。 发现6（R，S）-甲基四氢蝶呤在不存在BH 4的情况下提高纯化的酶中的NOS活性。然而，脱氮类似物6（R，S）-甲基-5-脱氮四氢蝶呤不仅不能支持酶的周转，而且还以浓度依赖性方式抑制瓜氨酸的形成。总的来说，这些结果支持BH 4在NOS反应中的作用，该反应涉及酶的稳定和氧化还原化学，其中结合的蝶呤和NOS亚基之间的1：1化学计量导致最大活性。
Nitric oxide synthase (NOS) (EC 1.14.23) catalyzes the oxidation of L-arginine to citrulline and nitric oxide. The complex reaction carried out by NOS, which involves NADPH, O2, and enzyme-bound FAD, FMN, and tetrahydrobiopterin (BH4), has only recently begun to be elucidated. Herein we report the characterization of the pterin requirement of murine macrophage NOS. Although purified NOS activity was not dependent on BH4, activity was significantly enhanced by BH4 in a concentration-dependent fashion. NOS purified in the absence of added BH4 was found to contain substoichiometric concentrations of enzyme-bound pterin, where increased concentrations of bound pterin correlated with an increase in activity when assayed in the absence of exogenous BH4. However, NOS purified in the presence of BH4 followed by gel filtration exhibited a 1 mol of pterin:1 mol of NOS 130-kDa subunit stoichiometry and activity that was essentially independent of exogenous BH4. Experiments to probe a redox role for the pterin were carried out using pterin analogues. 6(R,S)-Methyltetrahydropterin was found to increase NOS activity in enzyme purified in the absence of BH4. However, the deaza analogue, 6(R,S)-methyl-5-deazatetrahydropterin, was not only incapable of supporting enzymatic turnover but also inhibited citrulline formation in a concentration-dependent manner. Overall, these results support a role for BH4 in the NOS reaction that involves stabilization of the enzyme and redox chemistry wherein a 1:1 stoichiometry between bound pterin and NOS subunit results in maximum activity.