Allosteric regulation of neuronal nitric oxide synthase by tetrahydrobiopterin and suppression of auto-damaging superoxide
Allosteric regulation of neuronal nitric oxide synthase by tetrahydrobiopterin and suppression of auto-damaging superoxide
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DOI：
10.1042/0264-6021:3460767
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发表时间：
2000-03-15
影响因子：
4.1
通讯作者：
Schmidt, HHHW
中科院分区：
文献类型：
--
作者：
Kotsonis, P;Fröhlich, LG;Schmidt, HHHW
调节同型二聚体一氧化氮合酶（NOS）家族活性的潜在机制，特别是对（6 R）-5，6，7，8-四氢-L-生物蝶呤（H（4）Bip）的需求尚未完全了解。在此，我们研究了在底物L-精氨酸转化为L-瓜氨酸和一氧化氮的过程中，H（4）Bip对神经元NOS（NOS-I）可能的变构和稳定作用。事实上，在动力学研究中，L-精氨酸和H（4）Bip之间的双重变构相互作用激活重组人NOS-I以增加L-精氨酸周转。与此一致的是，H（4）Bip而不是基于蝶呤的NOS抑制剂2-氨基-4，6-二氧代-3，4，5，6，8，8a，9，10-八氢-恶唑并[1，2-f]-蝶啶（PHS-32）引起血红素Soret带中L-精氨酸依赖性增加，表明底物与重组人NOS-I的结合增加。相反，L-精氨酸以浓度依赖性方式增加H（4）Bip与猪脑NOS-I的结合。其次，我们研究了H（4）Bip对NOS四元结构的稳定作用与解偶联催化的关系。在催化测定条件下，在不存在H（4）Bip的情况下，二聚体重组人NOS-I解离成无活性单体。单体化与还原氧活化的解偶联有关，因为它被超氧化物歧化酶和抑制剂N-w-硝基-L-精氨酸抑制。重要的是，H（4）Bip被发现与超氧化物（O-2（-.））酶结合的H（4）Bip在O-2（-.）在不存在底物的情况下产生条件。这些结果表明，H（4）Bip通过直接拦截自损伤O-2（-.）的新机制，变构激活NOS-I并稳定四级结构。
The underlying mechanisms regulating the activity of the family of homodimeric nitric oxide synthases (NOSs) and, in particular, the requirement for (6R)-5,6,7,8-tetrahydro-L-biopterin (H(4)Bip) an not fully understood. Here we have investigated possible allosteric and stabilizing effects of H(4)Bip on neuronal NOS (NOS-I) during the conversion of substrate, L-arginine, into L-citrulline and nitric oxide. Indeed, in kinetic studies dual allosteric interactions between L-arginine and H(4)Bip activated recombinant human NOS-I to increase L-arginine turnover. Consistent with this was the observation that H(4)Bip, but not the pterin-based NOS inhibitor 2-amino-4,6-dioxo-3,4,5,6,8,8a,9,10-octahydro-oxazolo[1,2-f]-pteridine (PHS-32), caused an L-arginine-dependent increase in the haem Soret band, indicating an increase in substrate binding to recombinant human NOS-I. Conversely, L-arginine was observed to increase in a concentration-dependent manner H(4)Bip binding to pig brain NOS-I. Secondly, we investigated the stabilization of NOS quaternary structure by H(4)Bip in relation to uncoupled catalysis. Under catalytic assay conditions and in the absence of H(4)Bip, dimeric recombinant human NOS-I dissociated into inactive monomers. Monomerization was related to the uncoupling of reductive oxygen activation, because it was inhibited by both superoxide dismutase and the inhibitor N-w-nitro-L-arginine. Importantly, H(4)Bip was found to react chemically with superoxide (O-2(-.)) and enzyme-bound H(4)Bip was consumed under O-2(-.)-generating conditions in the absence of substrate. These results suggest that H(4)Bip allosterically activates NOS-I and stabilizes quaternary structure by a novel mechanism involving the direct interception of auto-damaging O-2(-.).