p.V132K Val132Lys (p.V163K Val163Lys in primary sequence with 31 amino-acids signal peptide) Back door hypothesis;V132K introduce a charge and should block the door, 6X increase of Km for ATc but not for TB through interaction with W86
Title: The back door hypothesis for product clearance in acetylcholinesterase challenged by site-directed mutagenesis Kronman C, Ordentlich A, Barak D, Velan B, Shafferman A Ref: Journal of Biological Chemistry, 269:27819, 1994 : PubMed
The active site of acetylcholinesterase is near the bottom of a long and narrow gorge. The dimensions of the gorge and the strong electrostatic field generated by the enzyme appear inconsistent with the enzyme's high turnover rate. Consequently, a "back door" mechanism involving movement of the reaction products through a transient opening near the active center was recently suggested. We investigated this hypothesis in human acetylcholinesterase by testing mutants at key residues (Glu-84, Trp-86, Asp-131, and Val-132) located near or along the putative back door channel. The turnover rates of all mutants tested, and in particular of V132K, where the channel is expected to be sealed by salt bridge Lys-132-Glu-452, are similar to that of the wild type enzyme. This indicates that the proposed back door is not a route for product clearance from the active site gorge of acetylcholinesterase and is probably of no functional relevance to its catalytic activity.
