Schallreuter Karin U, Elwary Souna M A, Gibbons Nicholas C J, Rokos Hartmut, Wood John M
Department of Biomedical Sciences, Clinical and Experimental Dermatology, University of Bradford, Bradford, West Yorkshire BD7 1DP, UK.
Biochem Biophys Res Commun. 2004 Mar 5;315(2):502-8. doi: 10.1016/j.bbrc.2004.01.082.
Previously it has been demonstrated that the human epidermis synthesises and degrades acetylcholine and expresses both muscarinic and nicotinic receptors. These cholinergic systems have been implicated in the development of the epidermal calcium gradient and differentiation in normal healthy skin. In vitiligo severe oxidative stress occurs in the epidermis of these patients with accumulation of H2O2 in the 10(-3)M range together with a decrease in catalase expression/activity due to deactivation of the enzyme active site. It was also shown that the entire recycling of the essential cofactor (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin via pterin-4a-carbinolamine dehydratase (PCD) and dihydropteridine reductase (DHPR) is affected by H2O2 oxidation of Trp/Met residues in the enzyme structure leading to deactivation of these proteins. Using fluorescence immunohistochemistry we now show that epidermal H2O2 in vitiligo patients yields also almost absent epidermal acetylcholinesterase (AchE). A kinetic analysis using pure recombinant human AchE revealed that low concentrations of H2O2 (10(-6)M) activate this enzyme by increasing the Vmax>2-fold, meanwhile high concentrations of H2O2 (10(-3)M) inhibit the enzyme with a significant decrease in Vmax. This result was confirmed by fluorescence excitation spectroscopy following the Trp fluorescence at lambdamax 280nm. Molecular modelling based on the established 3D structure of human AchE supported that H2O2-mediated oxidation of Trp(432), Trp(435), and Met(436) moves and disorients the active site His(440) of the enzyme, leading to deactivation of the protein. To our knowledge these results identified for the first time H2O2 regulation of AchE. Moreover, it was shown that H2O2-mediated oxidation of AchE contributes significantly to the well-established oxidative stress in vitiligo.
此前已有研究表明，人类表皮能够合成和降解乙酰胆碱，并表达毒蕈碱型和烟碱型受体。这些胆碱能系统与正常健康皮肤中表皮钙梯度的形成及分化有关。在白癜风患者中，表皮会出现严重的氧化应激，过氧化氢在表皮中的积累量达到10⁻³M范围，同时由于酶活性位点失活，过氧化氢酶的表达/活性降低。研究还表明，必需辅因子（6R）-l-赤藓糖-5,6,7,8-四氢生物蝶呤通过蝶呤-4a-甲醇胺脱水酶（PCD）和二氢蝶啶还原酶（DHPR）的整个循环过程会受到酶结构中色氨酸/甲硫氨酸残基被过氧化氢氧化的影响，从而导致这些蛋白质失活。现在我们通过荧光免疫组织化学方法发现，白癜风患者表皮中的过氧化氢还会导致表皮乙酰胆碱酯酶（AchE）几乎缺失。使用纯重组人AchE进行的动力学分析表明，低浓度的过氧化氢（10⁻⁶M）通过使最大反应速度（Vmax）增加两倍以上来激活该酶，而高浓度的过氧化氢（10⁻³M）则抑制该酶，使Vmax显著降低。在280nm波长处追踪色氨酸荧光的荧光激发光谱证实了这一结果。基于已建立的人AchE三维结构的分子模拟表明，过氧化氢介导的色氨酸（432）、色氨酸（435）和甲硫氨酸（436）的氧化会使酶的活性位点组氨酸（440）移动并使其取向紊乱，从而导致蛋白质失活。据我们所知，这些结果首次确定了过氧化氢对AchE的调节作用。此外，研究还表明，过氧化氢介导的AchE氧化对白癜风中已确定的氧化应激有显著贡献。
