Equilibrium in the hydrolysis and synthesis of cannabimimetic anandamide demonstrated by a purified enzyme

Anandamide, an endogenous ligand for cannabinoid receptors, loses its biological activities when it is hydrolyzed to arachidonic acid and ethanolamine by anandamide amidohydrolase. We overexpressed a recombinant rat enzyme with a hexahistidine tag in a baculovirus–insect cell expression system, and purified the enzyme with the aid of a Ni-charged resin to a specific activity as high as 5.7 μmol/min/mg protein. The purified recombinant enzyme catalyzed not only the hydrolysis of anandamide and palmitoylethanolamide, but also their reverse synthetic reactions. In order to attain an equilibrium of the anandamide hydrolysis and its reverse reaction within 10 min, we utilized a large amount of the purified enzyme. The equilibrium constant ([arachidonic acid][ethanolamine])/([anandamide][water]) was calculated as 4×10−3 (37°C, pH 9.0). These experimental results with a purified enzyme preparation quantitatively confirmed the reversibility of the enzyme reaction previously observed with crude enzyme preparations.