Importance of valine 567 in substrate recognition and oxidation by neuronal nitric oxide synthase.
Importance of valine 567 in substrate recognition and oxidation by neuronal nitric oxide synthase.
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DOI：
10.1016/j.jinorgbio.2004.03.015
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发表时间：
2004-07
影响因子：
3.9
通讯作者：
Magali Moreau;Hiroto Takahashi;M. Sari;J. Boucher;I. Sagami;Toru Shimizu;D. Mansuy
中科院分区：
文献类型：
--
作者：
Magali Moreau;Hiroto Takahashi;M. Sari;J. Boucher;I. Sagami;Toru Shimizu;D. Mansuy
一氧化氮（NO）是由一氧化氮合酶（NOS）活性部位的L-精氨酸（L-Arg）经两步氧化生成中间体Nω-羟基-L-精氨酸（NOHA）而合成的。NOS的晶体结构已经显示了活性位点Val 567残基（编号为大鼠神经元NOS，nNOS）与非氨基酸底物相互作用的重要性。为了研究该瓦尔残基在nNOS的底物识别和NO形成活性中的作用，我们产生并纯化了nNOS的四种Val 567突变体，Val 567 Leu，Val 567 Phe，Val 567 Arg和Val 567 Glu。我们表征了这些蛋白质，并测试了它们从天然底物L-Arg和NOHA的氧化以及从先前确定为nNOS的替代底物的N-羟基胍产生NO的能力。在所有测试化合物存在下，Val 567 Leu突变体显示出比野生型（WT）更低的NO形成活性。令人惊讶的是，Val 567 Phe突变体仅从NOHA形成少量的NO。这两个突变体显示出比WT蛋白更低的对l-Arg和NOHA的亲和力。Val 576 Glu和Val 567 Arg突变体的稳定性要低得多，并没有导致任何NO的形成。这些结果表明，Val 567是一个重要的残基，用于保持活性位点的完整性，底物结合，并随后在nNOS中形成NO。
Nitric oxide (NO) is synthesised by a two-step oxidation of l-arginine (l-Arg) in the active site of nitric oxide synthase (NOS) with formation of an intermediate, Nω-hydroxy-l-Arg (NOHA). Crystal structures of NOSs have shown the importance of an active-site Val567 residue (numbered for rat neuronal NOS, nNOS) interacting with non-amino acid substrates. To investigate the role of this Val residue in substrate recognition and NO-formation activity by nNOS, we generated and purified four Val567 mutants of nNOS, Val567Leu, Val567Phe, Val567Arg and Val567Glu. We characterized these proteins and tested their ability to generate NO from the oxidation of natural substrates l-Arg and NOHA, and from N-hydroxyguanidines previously identified as alternative substrates for nNOS. The Val567Leu mutant displayed lower NO formation activities than the wild type (WT) in the presence of all tested compounds. Surprisingly, the Val567Phe mutant formed low amounts of NO only from NOHA. These two mutants displayed lower affinity for l-Arg and NOHA than the WT protein. Val576Glu and Val567Arg mutants were much less stable and did not lead to any formation of NO. These results suggest that Val567 is an important residue for preserving the integrity of the active site, for substrate binding, and subsequently for NO-formation in nNOS.