Stone Everett M, Costello Alison L, Tierney David L, Fast Walter
Graduate Program in Cell and Molecular Biology, The University of Texas at Austin, Austin, Texas 78712, USA.
Biochemistry. 2006 May 2;45(17):5618-30. doi: 10.1021/bi052595m.
The enzyme dimethylargininase (also known as dimethylarginine dimethylaminohydrolase or DDAH; EC 3.5.3.18) catalyzes the hydrolysis of endogenous nitric oxide synthase inhibitors, N(omega)-methyl-l-arginine and N(omega),N(omega)-dimethyl-l-arginine. Understanding the mechanism and regulation of DDAH activity is important for developing ways to control nitric oxide production during angiogenesis and in many cases of vascular endothelial pathobiology. Several possible physiological regulation mechanisms of DDAH depend upon the presence of an active-site cysteine residue, Cys249 in Pseudomonas aeruginosa (Pa) DDAH, which is proposed to serve as a nucleophile in the catalytic mechanism. Through the use of pH-dependent ultraviolet and visible (UV-vis) difference spectroscopy and inactivation kinetics, the pK(a) of the active-site Cys249 in the resting enzyme was found to be unperturbed from pK(a) values of typical noncatalytic cysteine residues. In contrast, the pH dependence of k(cat) values indicates a much lower apparent pK(a) value. UV-vis difference spectroscopy between wild-type and C249S DDAH shows absorbance changes consistent with Cys249 deprotonation to the anionic thiolate upon binding positively charged ligands. The proton from Cys249 is lost either to the solvent or to an unidentified general base. A mutation of the active-site histidine residue, H162G, does not eliminate cysteine nucleophilicity, further arguing against a pre-formed ion pair with Cys249. Finally, UV-vis and X-ray absorption spectroscopy revealed that inhibitory metal ions can bind at these two active-site residues, Cys249 and His162, and also stabilize the anionic form of Cys249. These results support a proposed substrate-assisted mechanism for Pa DDAH in which ligand binding modulates the reactivity of the active-site cysteine.
二甲基精氨酸酶（也称为二甲基精氨酸二甲氨基水解酶或DDAH；EC 3.5.3.18）催化内源性一氧化氮合酶抑制剂N（ω）-甲基-L-精氨酸和N（ω），N（ω）-二甲基-L-精氨酸的水解。了解DDAH活性的机制和调节对于开发在血管生成过程中以及在许多血管内皮病理生物学情况下控制一氧化氮产生的方法很重要。DDAH的几种可能的生理调节机制取决于活性位点半胱氨酸残基的存在，铜绿假单胞菌（Pa）DDAH中的Cys249，它被认为在催化机制中作为亲核试剂。通过使用pH依赖性紫外可见（UV-vis）差分光谱和失活动力学，发现静息酶中活性位点Cys249的pK（a）与典型的非催化性半胱氨酸残基的pK（a）值没有扰动。相比之下，k（cat）值的pH依赖性表明表观pK（a）值要低得多。野生型和C249S DDAH之间的UV-vis差分光谱显示，在结合带正电荷的配体时，吸光度变化与Cys249去质子化为阴离子硫醇盐一致。来自Cys249的质子要么丢失到溶剂中，要么丢失到一个未确定的通用碱中。活性位点组氨酸残基H162G的突变不会消除半胱氨酸的亲核性，这进一步反对了与Cys249预先形成的离子对。最后，UV-vis和X射线吸收光谱显示，抑制性金属离子可以结合在这两个活性位点残基Cys249和His162上，并且还稳定Cys249的阴离子形式。这些结果支持了一种提出的Pa DDAH底物辅助机制，其中配体结合调节活性位点半胱氨酸的反应性。
