Cholinesterases

• Acetylcholinesterase locations:

  • Cholinergic nerve terminals
  • Motor end plates in muscle
  • Red blood cells
• Substrates of acetylcholinesterase:

  • Acetylcholine
  • Methacholine
• Poor substrates/no activity of acetylcholinesterase:

  • Carbachol
  • Benzylcholine
  • Suxamethonium
• Butyrylcholinesterase = Pseudo-/soluble cholinesterase
•  Butyrylcholinesterase has widespread distribution, including in:

  • Liver
  • Skin
  • Brain
  • GI smooth muscle
• Substrates of butyrylcholinesterase

  • Butyrylcholine
  • Acetylcholine
  • Benzylcholine
  • Suxamethonium
• Poor substrates/no activity of butyrylcholinesterase

  • Methacholine
  • Carbachol
• Butyrylcholine is essentially a larger version of acetylcholine (butyryl group instead of methyl group)
• Carbachol has an amine group in place of the methyl group
• Benzylcholine has an aromatic group in place of the methyl group
• Beta-methacholine has a methyl group on the beta carbon
• Suxamethonium is double the acetylcholine molecule
• Acetylcholinesterases are expressed in close proximity to ACh receptors, ensuring rapid degradation of ACh after activation
• Membrane bound AChE

  • Collagen-like triple helix structure anchors the protein in the membrane
  • Each branch is a separate enzyme
• Enzymes of AChE consists of four subunits
• The subunits of AChE are held together by disulphide bonds
• Butyrylcholinesterase is the soluble form of AChE
• Butrylcholinesterase lacks the collagen-like anchor and consists of just one enzyme
• Peripheral binding site = anionic binding site
• The peripheral binding site attracts ACh into the site
• The quaternary amine of ACh forms a pi-cation bond with glutamate in the peripheral binding site
• ACh forms a series of pi-cation interactions with phenylalanine and tryptophan to move through the gorge and into the active site
• The quaternary amine of ACh is stabilised by tryptophan via a pi-cation bond in the catalytic site
• Serine, histidine and glutamate residues form the catalytic triad
• catalytic site = active site
• The volume of the active site for butyrylcholinesterase is larger than for acetylcholinesterase to accommodate for larger substrates
• Step 1: Acylation

  1. H in histidine is attracted to the -OH of glutamate, causing the N of histidine to have a lone pair of electrons
  2. The lone pair of electrons attacks the -OH of serine, causing the O to become reactive
  3. The O of serine forms a covalent bond with the acetate of ACh, forming a tetrahedral intermediate
  4. One H on histidine returns to acetate to acylate the serine, releasing choline and regenerating histidine and glutamate
• Step 2: Deacylation

  1. The lone electrons of the N in histidine attack water
  2. The water attacks the acetate on the serine residue, causing acetic acid to be released and the active enzyme to be regenerated
• Physostigmine/eserine are reversible AChE inhibitors
• Physostigmine has a similar structure to ACh, but has a carbamate group
• Physostigmine forms a carbamylated form of the enzyme
• The duration of the inhibitor's action depends on the rate of hydrolysis of the carbamylated enzyme
• Donepezil is a non-competitive, reversible AChE inhibitor
• The aromatic groups of donepezil forms a number of pi-pi interactions in the gorge of the active site, but does not directly interact with the catalytic triad
• Organophosphates are organic molecules with a phosphate group
• Organophosphates are nerve agents and insecticides
• Organophosphates form a phosphorylated version of the enzyme, which is very strong and will not hydrolyse spontaneously
• Nerve agents

  • Sarin
  • VX
  • Novichok (mixture of molecules)
• In insecticides, the phosphate group has a double bond with a sulphur
• The phosphorus-sulphur double bond in insecticides means that it is inactive on mammalian acetylcholinesterases