Forbes Scott P, Druhan Lawrence J, Guzman Jorge E, Parinandi Narasimham, Zhang Liwen, Green-Church Kari B, Cardounel Arturo J
Department of Physiology and Functional Genomics, The University of Florida College of Medicine, Gainesville, Florida 32610, USA.
Biochemistry. 2008 Feb 12;47(6):1819-26. doi: 10.1021/bi701659n. Epub 2008 Jan 3.
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 l-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 Km values of 68.7 and 53.6 microM and Vmax 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 >or=100 microM 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 microM, 50% inhibition at 50 microM, and complete inhibition at 500 microM. 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.
一氧化氮合酶受到精氨酸的NG-甲基化衍生物的抑制，其细胞水平由二甲基精氨酸二甲胺水解酶（DDAH）控制。DDAH-1是一种含锌（II）的酶，通过甲基化L-精氨酸的水解来调节一氧化氮合酶的活性。在此，我们报告了人DDAH-1的动力学特性及其氧化还原依赖性调节。使用重组酶进行的动力学研究表明，对于非对称二甲基精氨酸（ADMA）和N-甲基-L-精氨酸（L-NMMA），其米氏常数（Km）值分别为68.7和53.6微摩尔，最大反应速度（Vmax）值分别为356和154纳摩尔/毫克/分钟。这种酶活性对游离的ADMA和L-NMMA具有选择性，并且不能水解肽结合的甲基精氨酸。随后进行的研究以确定活性氧和活性氮物质对DDAH活性的影响，结果表明低水平的氧化剂暴露对酶活性影响很小，需要接近或大于100微摩尔的浓度才能显著抑制DDAH活性。然而，将DDAH暴露于脂质氧化产物4-羟基壬烯醛（4-HNE）时，其活性呈剂量依赖性抑制，在10微摩尔时观察到15%的抑制，在50微摩尔时抑制50%，在500微摩尔时完全抑制。质谱分析表明，抑制机制是由于在位于人DDAH-1活性位点催化三联体中的组氨酸173上形成了迈克尔加成物。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一氧化氮合酶活性受损和一氧化氮生物利用度降低。
