Human epidermal acetylcholinesterase (AchE) is regulated by hydrogen peroxide (H 2 O 2 )

Previously it has been demonstrated that the human epidermis synthesizes 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 H 2 O 2 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‐erytho 5, 6, 7, 8 tetrahydrobiopterin via pterin‐4a‐carbinolamine dehydratase (PCD) and dihydropteridine reductase (DHPR) is affected by H 2 O 2 oxidation of Trp/Met residues in the enzyme structure leading to deactivation of these proteins. Using fluorescence immunohistochemistry we now show that epidermal H 2 O 2 in vitiligo patients yields also almost absent epidermal acetylcholinesterase (AchE) in association with accumulation of epidermal acetylcholine. This result was confirmed by Fluorescence excitation spectroscopy following the Trp fluorescence at λ max 280 nm. A kinetic analysis using pure recombinant human AchE revealed that low concentrations of H 2 O 2 (10 −6 M) activate this enzyme by increasing the Vmax > 2 fold, meanwhile high concentrations of H 2 O 2 (10 −3 M) deactivate the enzyme with a significant decrease in Vmax. Molecular modelling based on the established 3D structure of human AchE supported that H 2 O 2 ‐mediated oxidation of Trp 432 , Trp 435 and Met 436 moves and disorients the active site His 440 of the enzyme, thus explaining the deactivation of the protein. To our knowledge these results identified for the first time H 2 O 2 regulation of AchE. Moreover, it was shown that H 2 O 2 ‐mediated oxidation of AchE contributes significantly to the well established oxidative stress in vitiligo.