Polyproline-rich Peptides Organize 4 Cholinesterase Subunits into A Tetramer; BChE and AChE Scavenge Polyproline Peptides Released during Metabolic Turnover

Abstract

The genes for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) encode the proteins responsible for enzyme activity. Additional gene products, PRiMA and ColQ, anchor AChE and BChE proteins into membranes. Soluble AChE and BChE tetramers are composed of 4 identical subunits plus one polyproline-rich peptide. Dilution does not release the polyprolinerich peptide from tetramers. However, protein denaturation, for example heating in a boiling water bath, dissociates the polyproline-rich peptide. Using mass spectrometry to sequence peptides released from soluble AChE and BChE tetramers, we find sequences that correspond to proline-rich regions from a variety of proteins. A typical peptide sequence contains 20 consecutive prolines in a 23-residue peptide LPPPPPPPPPPPPPPPPPPPPLP. There is no single, common consensus sequence i.e., no specific gene appears to be responsible for the polyproline-rich peptides found in soluble AChE and BChE tetramers. We propose that during metabolic turnover, protein fragments containing polyproline-rich sequences are scavenged by AChE and BChE dimers, to make stable AChE and BChE tetramers. The 40-residue, alpha-helical C-terminus of AChE or BChE is the tetramerization domain that binds the polyproline-rich peptide. Four parallel alpha helices wrap around a single antiparallel polyproline peptide to lock the tetramer in place. This organization was established by classical X-ray crystallography for isolated C-termini in complex with a proline-rich peptide. The organization was confirmed for intact, tetrameric human BChE using cryoelectron microscopy. When 40 amino acids are deleted from the carboxy terminus, monomeric enzymes are created that retain full enzymatic activity.