Mechanism of 4-HNE mediated inhibition of hDDAH-1: Implications in no regulation
Mechanism of 4-HNE mediated inhibition of hDDAH-1: Implications in no regulation
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DOI：
10.1021/bi701659n
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发表时间：
2008-02-12
期刊：
影响因子：
2.9
通讯作者：
Cardounel, Arturo J.
中科院分区：
文献类型：
--
作者：
Forbes, Scott P.;Druhan, Lawrence J.;Cardounel, Arturo J.
一氧化氮合酶被精氨酸的NG-甲基化衍生物抑制，其细胞水平由二甲基精氨酸二甲氨基水解酶（DDAH）控制。DDAH-1是一种含Zn（II）的酶，其通过水解甲基化的1-精氨酸来调节NOS的活性。在此，我们报告的动力学特性的hDDAH-1和它的氧化还原依赖性调节。使用重组酶的动力学研究表明，ADMA和L-NMMA的Km值分别为68.7和53.6 μ M，V-max值分别为356和154 nmol/mg/min。这种酶活性对游离ADMA和L-NMMA具有选择性，并且不能水解掺入甲基精氨酸的肽。随后进行的确定活性氧和活性氮物质对DDAH活性的影响的研究表明，低水平的氧化剂暴露对酶活性几乎没有影响，并且需要接近>= 100 μ M的浓度来赋予DDAH活性的显著抑制。然而，DDAH暴露于脂质氧化产物4-HNE，剂量依赖性地抑制DDAH活性，在10 μ M时观察到15%的抑制，在50 μ M时观察到50%的抑制，在500 μ M时观察到完全抑制。质谱分析表明，抑制机制是由于His 173上形成Michael加合物所致，His 173位于hDDAH-1的活性位点催化三联体内。这些研究是在这种脂质过氧化产物的病理生理相关浓度下进行的，表明在氧化应激增加的条件下DDAH活性可能会受损。由于DDAH是参与甲基精氨酸代谢的主要酶，该酶活性的丧失将导致NOS活性受损和NO生物利用度降低。
Nitric oxide synthase is inhibited by NG-methylated derivatives of arginine whose cellular levels are controlled by dimethylarginine dimethylamino-hydrolase (DDAH). DDAH-1 is a Zn(II)containing enzyme that through hydrolysis of methylated 1-arginines regulates the activity of NOS. Herein, we report the kinetic properties of hDDAH-1 and its redox-dependent regulation. Kinetic studies using recombinant enzyme demonstrated K-m values of 68.7 and 53.6 mu M and V-max values of 356 and 154 nmols/mg/min for ADMA and L-NMMA, respectively. This enzymatic activity was selective for free ADMA and L-NMMA and was incapable of hydrolyzing peptide incorporated methylarginines. Subsequent studies performed to determine the effects of reactive oxygen and reactive nitrogen species on DDAH activity demonstrated that low level oxidant exposure had little effect on enzyme activity and that concentrations approaching >= 100 mu M were needed to confer significant inhibition of DDAH activity. However, exposure of DDAH to the lipid oxidation product, 4-HNE, dose-dependently inhibited DDAH activity with 15% inhibition observed at 10 mu M, 50% inhibition at 50 mu M, and complete inhibition at 500 mu M. Mass spectrometry analysis demonstrated that the mechanism of inhibition resulted from the formation of Michael adducts on His 173, which lies within the active site catalytic triad of hDDAH-1. These studies were performed with pathophysiologicaly relevant concentrations of this lipid peroxidation product and suggest that DDAH activity can be impaired under conditions of increased oxidative stress. Because DDAH is the primary enzyme involved in methylarginine metabolism, the loss of activity of this enzyme would result in impaired NOS activity and reduced NO bioavailability.