Tetrahydrobiopterin inhibits monomerization and is consumed during catalysis in neuronal NO synthase
Tetrahydrobiopterin inhibits monomerization and is consumed during catalysis in neuronal NO synthase
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DOI：
10.1074/jbc.274.35.24921
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发表时间：
1999-08-27
影响因子：
4.8
通讯作者：
Schmidt, HHHW
中科院分区：
文献类型：
--
作者：
Reif, A;Fröhlich, LG;Schmidt, HHHW
一氧化氮（NO）的生物合成是由同二聚体NO合成酶（NOS）催化的。由于未知的原因，所有NOS与亚化学计量量的（6 R）-5，6，7，8-四氢生物蝶呤（H（4）Bip）共纯化，并且需要额外的H（4）Bip以获得最大活性。我们研究了H（4）Bip和蝶呤衍生的抑制剂（antipterins）对纯化的神经元NOS-I四级结构和H（4）Bip含量的影响。在L-精氨酸周转过程中，NOS-I二聚体时间依赖性地解离成无活性单体，并伴随着酶相关蝶呤的丢失。当在催化过程中加入饱和水平的H（4）Bip时，二聚体的解离被抑制，在大肠杆菌中表达的无蝶呤的NOS-I也得到了类似的结果。H（4）Bip的这种稳定作用被抗蝶呤2-氨基-4，6-二氧代-3，4，5，6，8，8a，9，10-八氢-恶唑并[1，2f]-蝶啶（PHS-32）所模拟，其也以竞争性方式取代NOS相关的H（4）Bip。令人惊讶的是，H（4）Bip不仅在催化过程中从NOS解离，而且在溶质中仅部分回收（在20分钟时为对照的50.0 +/-16.5%）。NOS相关的H（4）Bip似乎与NOS催化产物反应生成不同于二氢生物蝶呤或生物蝶呤的衍生物。在相同条件下，试剂H（4）Bip化学稳定且完全回收（对照品的95.5 +/- 3.4%）。由精胺NONO-ate产生的NO观察到试剂和酶结合的H（4）Bip和二聚体含量的类似损失。总之，我们提出了一个作用，H（4）Bip作为二聚体稳定因子的神经元NOS在催化过程中，可能通过干扰酶的不稳定产物。
The biosynthesis of nitric oxide (NO) is catalyzed by homodimeric NO synthases (NOS), For unknown reasons, all NOS co-purify with substoichiometric amounts of (6R)-5,6,7,8-tetrahydrobiopterin (H(4)Bip) and require additional H(4)Bip for maximal activity. We examined the effects of H(4)Bip and pterin-derived inhibitors (antipterins) on purified neuronal NOS-I quaternary structure and H(4)Bip content. During L-arginine turnover, NOS-I dimers time dependently dissociated into inactive monomers, paralleled by a loss of enzyme-associated pterin. Dimer dissociation was inhibited when saturating levels of H(4)Bip were added during catalysis, Similar results were obtained with pterin-free NOS-I expressed in Escherichia coli. This stabilizing effect of H(4)Bip was mimicked by the anti-pterin 2-amino-4,6-dioxo-3,4,5,6,8,8a,9, 10-octahydro-oxazolo [1,2f]-pteridine (PHS-32), which also displaced NOS-associated H(4)Bip in a competitive manner. Surprisingly, H(4)Bip not only dissociated from NOS during catalysis, but was only partially recovered in the solute (50.0 +/- 16.5% of control at 20 min). NOS-associated H(4)Bip appeared to react with a NOS catalysis product to a derivative distinct from dihydrobiopterin or biopterin. Under identical conditions, reagent H(4)Bip was chemically stable and fully recovered (95.5 +/- 3.4% of control). A similar loss of both reagent and enzyme-bound H(4)Bip and dimer content was observed by NO generated from spermine NONO-ate. In conclusion, we propose a role for H(4)Bip as a dimer-stabilizing factor of neuronal NOS during catalysis, possibly by interfering with enzyme destabilizing products.