Anionic site interactions in human butyrylcholinesterase disrupted by two single point mutations

Area of Science:

• Biochemistry
• Molecular Biology
• Enzyme Kinetics

Background:

• Butyrylcholinesterase (CHE) is a key enzyme in neurotransmission and drug metabolism.
• Understanding CHE structure-function relationships is vital for developing targeted therapeutics and understanding enzyme activity.

Purpose of the Study:

• To elucidate the role of specific amino acid residues (Asp-70 and Ser-425) in the structure-function dynamics of recombinant human butyrylcholinesterase (CHE).
• To investigate how mutations at these sites affect ligand binding and inhibition properties.

Main Methods:

• Site-directed mutagenesis was employed to create CHE variants with specific amino acid substitutions (Ser-425 to Pro-425 and Asp-70 to Gly-70).
• Xenopus oocyte microinjection was used to express these recombinant human CHE variants.
• Ligand binding assays were performed using various substrates and inhibitors, including succinylcholine and dibucaine.

Main Results:

• The Ser-425 to Pro-425 mutation did not alter ligand binding affinity.
• The Asp-70 to Gly-70 substitution significantly diminished CHE's binding capacity for succinylcholine and specific inhibitors.
• Mutations at both sites conferred complete resistance to succinylcholine and dibucaine inhibition, while other substrate bindings remained unaffected.

Conclusions:

• Aspartic acid at position 70 (Asp-70) is a critical component of the anionic site for specific ligand interactions in human CHE.
• Structural interplay between Asp-70 and Ser-425 regions is suggested, potentially involving other electronegative amino acids in the anionic site binding.
• These findings provide insights into the molecular mechanisms underlying CHE activity and inhibition.