Metabolism of aminoguanidine, diaminoguanidine, and NG-amino-L-arginine by neuronal NO-synthase and covalent alteration of the heme prosthetic group.
Metabolism of aminoguanidine, diaminoguanidine, and NG-amino-L-arginine by neuronal NO-synthase and covalent alteration of the heme prosthetic group.
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DOI：
10.1021/tx050263c
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发表时间：
2005-12
影响因子：
4.1
通讯作者：
Anthony J. Lee;Kathleen R. Noon;S. Jianmongkol;Miranda Lau;Gary J. Jenkins;Y. Osawa
中科院分区：
文献类型：
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作者：
Anthony J. Lee;Kathleen R. Noon;S. Jianmongkol;Miranda Lau;Gary J. Jenkins;Y. Osawa
氨基胍（AG）、二氨基胍（DAG）和ng -氨基-l-精氨酸（NAA）是一氧化氮合酶（NOS）三种主要亚型的代谢灭活剂。在神经元NOS （nNOS）的情况下，已知血红素改变是失活的主要原因，尽管发生这种情况的确切机制尚不清楚。通过LC/MS/MS技术，我们发现AG、DAG和NAA在m/z分别为45.2、60.2和160.0时被nNOS代谢为相应质量离子的产物。这些结果与每种灭活剂的联氨部分的损失是一致的。这些发现通过精确的质量测量和对NAA和AG产品的真实标准的比较得到证实。AG、DAG和NAA在nNOS失活过程中形成的主要可解离血红素产物的m/z离子分别为660.2、675.2和775.3。这些结果与血红素加合物和失活剂减去肼部分一致。为了支持这一点，MS/MS研究在每种情况下都揭示了血红素的片段离子。通过使用14c标记的血红素，我们还表明，在AG的情况下，可解离血红素加合物约占改变的血红素的一半。此外，我们采用了一种基于软件的差异代谢分析方法，通过从不含nNOS的样品中减去含有nNOS的样品的LC/MS数据集来搜索复杂反应混合物中的产物和底物。本研究建立的代谢谱分析方法可作为搜索酶系统底物和产物的通用工具，包括药物代谢肝微粒体P450细胞色素。我们提出AG、DAG和NAA对nNOS的代谢失活是通过氧化去除肼基团和形成自由基中间体发生的，该中间体在h原子提取后形成稳定的产物或与血红素假体部分反应并使nNOS失活。
It is established that aminoguanidine (AG), diaminoguanidine (DAG), and NG-amino-l-arginine (NAA) are metabolism-based inactivators of the three major isoforms of nitric oxide synthase (NOS). In the case of neuronal NOS (nNOS), heme alteration is known to be a major cause of inactivation, although the exact mechanism by which this occurs is not well-understood. We show here by the use of LC/MS/MS techniques that AG, DAG, and NAA are metabolized by nNOS to products with corresponding mass ions at m/z of 45.2, 60.2, and 160.0, respectively. These results are consistent with the loss of a hydrazine moiety from each inactivator. These findings are confirmed by exact mass measurements and comparison to authentic standards in the case of the products for NAA and AG, respectively. Moreover, the major dissociable heme product that was formed during inactivation of nNOS by AG, DAG, and NAA had molecular ions at m/z 660.2, 675.2, and 775.3, respectively. These results are consistent with an adduct of heme and inactivator minus a hydrazine moiety. In support of this, MS/MS studies reveal a fragment ion of heme in each case. With the use of 14C-labeled heme, we also show that in the case of AG, the dissociable heme adduct accounts for approximately one-half of the heme that is altered. In addition, we employ a software-based differential metabolic profiling method by subtracting LC/MS data sets derived from samples that contained nNOS from those that did not contain the enzyme to search for products and substrates in complex reaction mixtures. The metabolic profiling method established in this study can be used as a general tool to search for substrates and products of enzyme systems, including the drug-metabolizing liver microsomal P450 cytochromes. We propose that the metabolism-based inactivation of nNOS by AG, DAG, and NAA occurs through oxidative removal of the hydrazine group and the formation of a radical intermediate that forms stable products after H-atom abstraction or reacts with the heme prosthetic moiety and inactivates nNOS.