Kotsonis P, Frey A, Fröhlich L G, Hofmann H, Reif A, Wink D A, Feelisch M, Schmidt H H
Department of Pharmacology and Toxicology, Julius-Maximilians University, Versbacher Strasse 9, D-97078 Würzburg, Germany.
Biochem J. 1999 Jun 15;340 ( Pt 3)(Pt 3):745-52.
Nitric oxide (NO) synthases (NOSs), which catalyse the oxidation of L-arginine to L-citrulline and an oxide of nitrogen, possibly NO or nitroxyl (NO-), are subject to autoinhibition by a mechanism that has yet to be fully elucidated. In the present study we investigated the actions of NO and other NOS-derived products as possible autoregulators of enzyme activity. With the use of purified NOS-I, L-arginine turnover was found to operate initially at Vmax (0-15 min, phase I) although, despite the presence of excess substrate and cofactors, prolonged catalysis (15-90 min, phase II) was associated with a rapid decline in L-arginine turnover. Taken together, these observations suggested that one or more NOS products inactivate NOS. Indeed, exogenously applied reactive nitrogen oxide species (RNSs) decreased Vmax during phase I, although with different potencies (NO->NO> ONOO-) and efficacies (NO>NO-=ONOO-). The NO scavengers oxyhaemoglobin (HbO2; 100 microM) and 1H-imidazol-1 - yloxy - 2 - (4-carboxyphenyl) - 4,5 - dihydro - 4,4,5,5 - tetramethyl - 3 -oxide (CPTIO; 10 microM) and the ONOO- scavenger GSH (7 mM) had no effect on NOS activity during phase I, except for an endogenous autoinhibitory influence of NO and ONOO-. However, superoxide dismutase (SOD; 300 units/ml), which is thought either to increase the half-life of NO or to convert NO- to NO, lowered Vmax in an NO-dependent manner because this effect was selectively antagonized by HbO2 (100 microM). This latter observation demonstrated the requirement of SOD to reveal endogenous NO-mediated autoinhibition. Importantly, during phase II of catalysis, NOS became uncoupled and began to form H2O2 because catalase, which metabolizes H2O2, increased enzyme activity. Consistent with this, exogenous H2O2 also inhibited NOS activity during phase I. Thus during catalysis NOS is subject to complex autoinhibition by both enzyme-derived RNS and H2O2, differentially affecting enzyme activity.
一氧化氮（NO）合酶（NOSs）催化L-精氨酸氧化为L-瓜氨酸和一种氮氧化物，可能是NO或硝酰基（NO-），其受到一种尚未完全阐明的机制的自身抑制作用。在本研究中，我们研究了NO和其他NOS衍生产物作为酶活性可能的自动调节因子的作用。使用纯化的NOS-I，发现L-精氨酸周转率最初以最大速度（Vmax）运行（0 - 15分钟，阶段I），尽管存在过量的底物和辅因子，但长时间催化（15 - 90分钟，阶段II）与L-精氨酸周转率的快速下降有关。综合这些观察结果表明，一种或多种NOS产物使NOS失活。实际上，外源性应用的活性氮氧化物（RNSs）在阶段I期间降低了Vmax，尽管具有不同的效力（NO->NO> ONOO-）和效果（NO>NO-=ONOO-）。NO清除剂氧合血红蛋白（HbO2；100 microM）和1H-咪唑-1 - 基氧基 - 2 - （4-羧基苯基） - 4,5 - 二氢 - 4,4,5,5 - 四甲基 - 3 - 氧化物（CPTIO；10 microM）以及ONOO-清除剂谷胱甘肽（GSH；7 mM）在阶段I期间对NOS活性没有影响，除了NO和ONOO-的内源性自身抑制作用。然而，超氧化物歧化酶（SOD；300单位/毫升），其被认为要么增加NO的半衰期，要么将NO-转化为NO，以NO依赖的方式降低了Vmax，因为这种作用被HbO2（100 microM）选择性拮抗。后一观察结果证明了SOD揭示内源性NO介导的自身抑制作用的必要性。重要的是，在催化的阶段II期间，NOS解偶联并开始形成H2O2，因为代谢H2O2的过氧化氢酶增加了酶活性。与此一致，外源性H2O2在阶段I期间也抑制了NOS活性。因此，在催化过程中，NOS受到酶衍生的RNS和H2O2的复杂自身抑制作用，对酶活性产生不同影响。
