Division of Molecular Structure and ‡Division of Physical Biochemistry, MRC National Institute for Medical Research , The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom.
Biochemistry. 2014 Feb 18;53(6):1092-104. doi: 10.1021/bi4015924. Epub 2014 Feb 7.
The dimethylarginine dimethylaminohydrolase (DDAH) enzyme family has been the subject of substantial investigation as a potential therapeutic target for the regulation of vascular tension. DDAH enzymes catalyze the conversion of asymmetric N(η),N(η)-dimethylarginine (ADMA) to l-citrulline. Here the influence of substrate and product binding on the dynamic flexibility of DDAH from Pseudomonas aeruginosa (PaDDAH) has been assessed. A combination of heteronuclear NMR spectroscopy, static and time-resolved fluorescence measurements, and atomistic molecular dynamics simulations was employed. A monodisperse monomeric variant of the wild-type enzyme binds the reaction product l-citrulline with a low millimolar dissociation constant. A second variant, engineered to be catalytically inactive by substitution of the nucleophilic Cys249 residue with serine, can still convert the substrate ADMA to products very slowly. This PaDDAH variant also binds l-citrulline, but with a low micromolar dissociation constant. NMR and molecular dynamics simulations indicate that the active site "lid", formed by residues Gly17-Asp27, exhibits a high degree of internal motion on the picosecond-to-nanosecond time scale. This suggests that the lid is open in the apo state and allows substrate access to the active site that is otherwise buried. l-Citrulline binding to both protein variants is accompanied by an ordering of the lid. Modification of PaDDAH with a coumarin fluorescence reporter allowed measurement of the kinetic mechanism of the PaDDAH reaction. A combination of NMR and kinetic data shows that the catalytic turnover of the enzyme is not limited by release of the l-citrulline product. The potential to develop the coumarin-PaDDAH adduct as an l-citrulline sensor is discussed.
二甲基精氨酸二甲胺水解酶（DDAH）酶家族一直是作为调节血管张力的潜在治疗靶点的大量研究的主题。DDAH 酶催化非对称 N(η),N(η)-二甲基精氨酸 (ADMA) 向 l-瓜氨酸的转化。在这里，评估了底物和产物结合对铜绿假单胞菌（PaDDAH）DDAH 动态灵活性的影响。采用异核 NMR 光谱、静态和时间分辨荧光测量以及原子分子动力学模拟的组合。野生型酶的单分散单体变体与反应产物 l-瓜氨酸结合具有低毫摩尔离解常数。通过用丝氨酸取代亲核 Cys249 残基工程改造的第二个变体仍能非常缓慢地将底物 ADMA 转化为产物。该 PaDDAH 变体也与 l-瓜氨酸结合，但离解常数为低微摩尔。NMR 和分子动力学模拟表明，由残基 Gly17-Asp27 形成的活性部位“盖子”在皮秒到纳秒的时间尺度上表现出高度的内部运动。这表明盖子在apo 状态下是打开的，允许底物进入否则被埋藏的活性部位。两种蛋白质变体的 l-瓜氨酸结合都伴随着盖子的有序化。用香豆素荧光报告器修饰 PaDDAH 允许测量 PaDDAH 反应的动力学机制。NMR 和动力学数据的组合表明，酶的催化周转率不受 l-瓜氨酸产物释放的限制。讨论了将香豆素-PaDDAH 加合物开发为 l-瓜氨酸传感器的潜力。
