Expanding cholinesterase function: kinetic and structural evidence for hydrolysis of long-chain fatty-acid esters

Cholinesterases (ChEs), including acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), are classically known for hydrolyzing choline esters in neurotransmission. However, their structural similarity to lipases suggests a possible capacity to act on lipid esters, implicating them in systemic lipid metabolism. The hydrolytic activity of human AChE (huAChE), purified human plasma BChE (huBChE), and equine serum BChE (EqBChE) was assayed with against three fluorescent fatty-acid esters-4-methylumbelliferyl palmitate (4-MU PA), 4-methylumbelliferyl oleate (4-MU OA), and 7-hydroxycoumarinyl arachidonate (7-HC AA) as substrate-using porcine pancreatic lipase (PPL) and human recombinant lipase (HRL) as positive controls. Kinetic parameters (Kₘ, Vₘₐₓ) were determined by Michaelis-Menten analysis. Enzyme specificity was assessed via inhibition by BW284C51 or iso-OMPA and lectin affinity assays. Molecular docking explored fatty-acid esters binding to ChE active sites. PPL and HRL hydrolyzed all substrates efficiently (Vₘₐₓ/Kₘ > 500 µU·mg⁻¹·µM⁻¹; Kₘ in the low-µM range). EqBChE and huBChE showed moderate activity (Vₘₐₓ/Kₘ ≈ 2-170), whereas huAChE displayed minimal activity, limited to 7-HC AA (Vₘₐₓ/Kₘ ≈ 1.4). Inhibitor pretreatment significantly reduced ChE activity (except for huAChE with 7-HC-AA), and concanavalin A retained < 10% of enzyme activity (p < 0.0001), confirming enzyme-dependent catalysis. Docking supported energetically favorable binding of lipid esters within ChE active sites. BChE demonstrates notable esterase activity toward long-chain fatty acid esters, whereas huAChE activity is largely selective for arachidonic acid derivatives. This atypical substrate preference implies a potential role for huAChE in arachidonic acid-linked inflammatory signaling, warranting further in vivo investigation.