Subunit Association and Stabilization of Butyrylcholinesterase (BChE)

Abstract

The goal of this project was to determine what parts of the BChE protein are important for its tetrameric organization. In human serum the BChE molecule has a dimer of dimers structure. Dimers are covalently linked by an interchain disulfide bond between the cysteines at amino acid 571. To study the effect the disulfide bond has on tetrameric structure and stability, a recombinant mutant was engineered replacing cysteine 571 with alanine. The mutant was expressed in Chinese hamster ovary cells. Elimination of the interchain disulfide bond at amino acid 571 decreased the heat stability of the enzyme but preserved its tetrameric structure. A second mutant in which 40 amino acids were deleted from the carboxy terminus was also expressed. This mutant gave an active monomer, thus suggesting that the region involved in dimer and tetramer contact is the carboxy terminus. Additional mutations, engineered by PCR (Polymerase Chain Reaction) site directed mutagenesis, will allow further characterization of the tetrameric stabilization of BChE.

The carboxy terminus, believed to be the contact point for dimer and tetramer formation, is depicted as a helix bundle with the interchain disulfide bond at Cys 571 stabilizing the dimers. We hypothesize that with proteolysis the carboxy terminal tail is cleaved and the subunits dissociate.