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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• 作者：Anthony J. Lee ; Kathleen R. Noon ; Suree Jianmongkol ; Miranda Lau ; Gary J. Jenkins ; and Yoichi Osawa
• 刊名：Chemical Research in Toxicology
• 出版年：2005
• 出版时间：December 2005
• 年：2005
• 卷：18
• 期：12
• 页码：1927 - 1933
• 全文大小：147K
• 年卷期：v.18,no.12(December 2005)
• ISSN：1520-5010

文摘

It is established that aminoguanidine (AG), diaminoguanidine (DAG), and N^G-amino-L-arginine (NAA) are metabolism-based inactivators of the three major isoforms of nitric oxidesynthase (NOS). In the case of neuronal NOS (nNOS), heme alteration is known to be a majorcause 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 metabolizedby 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. Thesefindings are confirmed by exact mass measurements and comparison to authentic standardsin the case of the products for NAA and AG, respectively. Moreover, the major dissociableheme product that was formed during inactivation of nNOS by AG, DAG, and NAA hadmolecular ions at m/z 660.2, 675.2, and 775.3, respectively. These results are consistent withan adduct of heme and inactivator minus a hydrazine moiety. In support of this, MS/MS studiesreveal a fragment ion of heme in each case. With the use of ¹⁴C-labeled heme, we also showthat in the case of AG, the dissociable heme adduct accounts for approximately one-half of theheme that is altered. In addition, we employ a software-based differential metabolic profilingmethod by subtracting LC/MS data sets derived from samples that contained nNOS from thosethat did not contain the enzyme to search for products and substrates in complex reactionmixtures. The metabolic profiling method established in this study can be used as a generaltool to search for substrates and products of enzyme systems, including the drug-metabolizingliver microsomal P450 cytochromes. We propose that the metabolism-based inactivation ofnNOS by AG, DAG, and NAA occurs through oxidative removal of the hydrazine group andthe formation of a radical intermediate that forms stable products after H-atom abstractionor reacts with the heme prosthetic moiety and inactivates nNOS.