Alteration of the heme prosthetic group of neuronal nitric-oxide synthase during inactivation by N(G)-amino-L-arginine in vitro and in vivo.
Alteration of the heme prosthetic group of neuronal nitric-oxide synthase during inactivation by N(G)-amino-L-arginine in vitro and in vivo.
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DOI：
10.1124/mol.62.1.110
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发表时间：
2002-07
影响因子：
3.6
通讯作者：
J. Vuletich;E. Lowe;S. Jianmongkol;Y. Kamada;U. Kent;Andrew T. Bender;Damon R. Demady;P. Hollenberg;Y. Osawa
中科院分区：
文献类型：
--
作者：
J. Vuletich;E. Lowe;S. Jianmongkol;Y. Kamada;U. Kent;Andrew T. Bender;Damon R. Demady;P. Hollenberg;Y. Osawa
已确定 N(G)-氨基-L-精氨酸 (NAA) 是所有三种主要一氧化氮合酶 (NOS) 亚型的基于代谢的灭活剂。然而，这种失活发生的机制尚不清楚。在当前的研究中，我们发现 NAA 体外灭活一氧化氮合酶 (nNOS) 的神经元同工型会导致血红素辅基发生共价改变，部分地变成含有完整卟啉环的产物，并且可以从蛋白质上解离或不可逆地结合到蛋白质上。血红素的改变伴随着 nNOS 活性的丧失。对含有 14C 标记的假体血红素部分的 nNOS 的研究表明，主要的可解离产物和不可逆结合的血红素加合物分别占被改变的血红素的 21% 和 28%。主要可解离产物的质谱分析给出了 m/z 775.3 的分子离子，该分子离子与血红素和 NAA 减去肼基团的加合物的质量一致。不可逆结合的血红素加合物的肽图表明，血红素与 nNOS 加氧酶结构域中的残基结合。我们首次证明，随着高度相似的血红素产物的形成，体内 nNOS 会发生基于代谢的失活。由于失活和改变可能引发 nNOS 的泛素化和蛋白酶体降解，因此 NAA 可能是研究这些基本调节过程的有用生化工具。
It is established that N(G)-amino-L-arginine (NAA) is a metabolism-based inactivator of all three major nitric-oxide synthase (NOS) isoforms. The mechanism by which this inactivation occurs, however, is not well understood. In the current study, we discovered that inactivation of the neuronal isoform of NOS (nNOS) by NAA in vitro results in covalent alteration of the heme prosthetic group, in part, to products that contain an intact porphyrin ring and are either dissociable from or irreversibly bound to the protein. The alteration of the heme is concomitant with the loss of nNOS activity. Studies with nNOS containing a 14C-labeled prosthetic heme moiety indicate that the major dissociable product and the irreversibly bound heme adduct account for 21 and 28%, respectively, of the heme that is altered. Mass spectral analysis of the major dissociable product gave a molecular ion of m/z 775.3 that is consistent with the mass of an adduct of heme and NAA minus a hydrazine group. Peptide mapping of the irreversibly bound heme adduct indicates that the heme is bound to a residue in the oxygenase domain of nNOS. We show for the first time that metabolism-based inactivation of nNOS occurs in vivo as highly similar heme products are formed. Because inactivation and alteration may trigger ubiquitination and proteasomal degradation of nNOS, NAA may be a useful biochemical tool for the study of these basic regulatory processes.