THE BACK DOOR HYPOTHESIS FOR PRODUCT CLEARANCE IN ACETYLCHOLINESTERASE CHALLENGED BY SITE-DIRECTED MUTAGENESIS

The active site of acetylcholinesterase is near the bottom of a long a nd narrow gorge. The dimensions of the gorge and the strong electrosta tic field generated by the enzyme appear inconsistent with the enzyme' s high turnover rate. Consequently, a ''back door'' mechanism involvin g movement of the reaction products through a transient opening near t he active center was recently suggested. We investigated this hypothes is in human acetylcholinesterase by testing mutants at key residues (G lu-84, Trp-86, Asp-131, and Val-132) located near or along the putativ e back door channel. The turnover rates of all mutants tested, and in particular of V132K, where the channel is expected to be sealed by sal t 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 p robably of no functional relevance to its catalytic activity.