TITLE:
Microcalorimetric Study of Acetylcholine and Acetylthiocholine Hydrolysis by Acetylcholinesterase
AUTHORS:
Paulo Alexandre A. de Almeida Neves, Eliane Novato Silva, Paulo S. L. Beirão
KEYWORDS:
Acetylcholinesterase, Acetylcholine, Acetylthiocholine, Isothermal Titration Calorimetry, Carbaryl
JOURNAL NAME:
Advances in Enzyme Research,
Vol.5 No.1,
March
9,
2017
ABSTRACT: Acetylcholinesterase (AChE) is an important enzyme responsible for the cleavage of acetylcholine. Studies of the activity of this enzyme use an artificial substrate, acetylthiocholine, because a product of its catalysis, thiocholine, readily generates a light absorbing product upon reaction with Elman’s reagent 5,5’-dithiobis-(2-nitrobenzoic acid (DTNB). The hydrolysis of acetylcholine cannot be assayed with this method. The isothermal titration calorimetry can assay the hydrolysis of both substrates, without requiring additional reagents other than the enzyme and the substrate. To compare kinetic values obtained in the hydrolysis of acetylcholine (ACh) and acetylthiocholine (ATCh), with carbaryl acting as inhibitor, a calorimetric technique was used to evaluate kinetic properties of the two reactions. This method can show the hydrolysis of both substrates by the heat exchange that occurs during catalysis. In addition, it allowed the assessment of the AChE inhibition by carbaryl, a common insecticide. The results show a similarity between values obtained with both substrates, which are slightly higher for acetylcholine, the enzyme natural substrate. Enzymatic parameters values from ATCh and ACh were similar to each other and inhibitory constants using carbaryl were also similar, displaying that any approach to ACh is feasible using ATCh. The results obtained from ITC show the precision achieved by the calorimetric method.