TETRAMERIZATION DOMAIN OF HUMAN BUTYRYLCHOLINESTERASE IS AT THE C-TERMINUS

Butyrylcholinesterase (BChE) in human serum consists predominantly of tetramers. Recombinant BChE, however, expressed in Chinese hamster ova ry (CHO) cells, consists of approx. 55% dimers, 10-30% tetramers and 1 5-40% monomers. To determine the origin of the monomer species we adde d the FLAG epitope (epitope tag, amino acid sequence DYKDDDDK) to the C-terminus of the enzyme, and expressed BChE-FLAG in CHO cells. We fou nd that secreted, active monomers had lost their FLAG epitope, suggest ing that the monomers were made by proteolysis of dimers or tetramers at the C-terminus. To estimate the number of amino acids that could be deleted from the C-terminus without losing BChE activity, we expresse d deletion mutants. We found that deletion of up to 50 amino acids fro m the C-terminus yielded active monomers, but that deletion of 51 amin o acids destroyed BChE activity and caused the inactive protein to rem ain within the cell. Deletion of eight or more amino acids from the N- terminus also resulted in inactive protein that remained inside the ce ll. Monomeric BChE had wild-type K-m and K-cat values (8 mu M and 2400 0 min(-1) for butyrylthiocholine) and showed substrate activation. The Cys-571-->Ala mutant, though incapable of forming the interchain disu lphide bond, had nearly the same amount of tetrameric BChE as recombin ant wild-type BChE. These results support the conclusion that the tetr amerization domain of BChE is at the C-terminus, within the terminal 5 0 amino acids, and that the interchain disulphide bond is not essentia l for tetramerization. Molecular modelling suggested that the tetramer ization domain was a four-helix bundle, stabilized by interactions of seven conserved aromatic amino acids.