Characterization of a transient covalent adduct formed during dimethylarginine dimethylaminohydrolase catalysis
Characterization of a transient covalent adduct formed during dimethylarginine dimethylaminohydrolase catalysis
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DOI：
10.1021/bi047407r
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发表时间：
2005-05-10
期刊：
影响因子：
2.9
通讯作者：
Fast, W
中科院分区：
文献类型：
--
作者：
Stone, EA;Person, MD;Fast, W
二甲基精氨酸二甲氨基水解酶（DDAH）调节人内源性一氧化氮合酶抑制剂N-omega-甲基- l-精氨酸（NMMA）和不对称N-omega，N-omega-二甲基- l-精氨酸（ADMA）的浓度。一氧化氮合成的药理调控是一个重要的目标，但DDAH的催化机制仍然很大程度上未被探索。从铜绿假单胞菌中克隆了一株DDAH，发现不对称甲基化精氨酸类似物是首选底物，ADMA的k(cat)/ k - m值略高于NMMA。DDAH类似于一个更大的胍基修饰酶超家族的成员，其中一些已被证明在催化过程中使用s -烷基硫脲中间体。在DDAH与NMMA或ADMA的稳态转换反应中未发现共价中间体积累。然而，鉴定出具有活化离去基的新底物s -甲基- l-硫辛瓜氨酸（SMTC），使酸捕获和ESI-MS表征了在稳态转换过程中积累的质量为158 +/- 10 Da的瞬态共价加合物。随后的捕获、蛋白水解、多肽定位和质谱裂解以及位点定向诱变表明，这种共价加合物附着在一个活性位点残基上，表明Cys249是中间产物形成所需的催化亲核试剂。共价催化的使用清楚地将DDAH与该酶超家族联系起来，并表明s -烷基硫脲中间体可能是其机制的保守特征。
Dimethylarginine dimethylaminohydrolase (DDAH) regulates the concentrations of human endogenous inhibitors of nitric oxide synthase, N-omega-methyl-L-arginine (NMMA), and asymmetric N-omega,N-omega-dimethyl-L-arginine (ADMA). Pharmacological regulation of nitric oxide synthesis is an important goal, but the catalytic mechanism of DDAH remains largely unexplored. A DDAH from Pseudomonas aeruginosa was cloned, and asymmetrically methylated arginine analogues were shown to be the preferred substrates, with ADMA displaying a slightly higher k(cat)/K-M value than NMMA. DDAH is similar to members of a larger superfamily of guanidino-modifying enzymes, some of which have been shown to use an S-alkylthiouronium intermediate during catalysis. No covalent intermediates were found to accumulate during steady-state turnover reactions of DDAH with NMMA or ADMA. However, identification of a new substrate with an activated leaving group, S-methyl-L-thiocitrulline (SMTC), enabled acid trapping and ESI-MS characterization of a transient covalent adduct with a mass of 158 +/- 10 Da that accumulates during steady-state turnover. Subsequent trapping, proteolysis, peptide mapping and fragmentation by mass spectrometry, and site-directed mutagenesis demonstrated that this covalent adduct was attached to an active site residue and implicates Cys249 as the catalytic nucleophile required for intermediate formation. The use of covalent catalysis clearly links DDAH to this superfamily of enzymes and suggests that an S-alkylthiouronium intermediate may be a conserved feature in their mechanisms.