Effects of mutations of active site residues and amino acids interacting with the Omega loop on substrate activation of butyrylcholinesterase

The peripheral anionic site (PAS) of human butyrylcholinesterase is involve d in the mechanism of substrate activation by positively charged substrates and ligands. Two substrate binding loci, D70 in the PAS and W82 in the act ive site, are connected by the Omega loop. To determine whether the Omega l oop plays a role in the signal transduction between the PAS and the active site, residues involved in stabilization of the loop, N83, K339 and W430, w ere mutated. Mutations N83A and N83Q caused loss of substrate activation, s uggesting that N83 which interacts with the D70 backbone may be an clement of the transducing system. The K339M and W430A mutant enzymes retained subs trate activation. Residues W82, E197, and A328 in the active site gorge hav e been reported to be involved in substrate activation. At butyrylthiocholi ne concentrations greater then 2 mM, W82A showed apparent substrate activat ion. Mutations E197Q and E197G strongly reduced substrate activation, while mutation E197D caused a moderate effect, suggesting that the carboxylate o f residue E197 is involved in substrate activation. Mutations A328F and A32 8Y showed no substrate activation, whereas A328G retained substrate activat ion. Substrate activation can result from an allosteric effect due to bindi ng of the second substrate molecule on the PAS. Mutation W430A was of speci al interest because this residue hydrogen bonds to W82 and Y332. W430A had strongly reduced affinity for tetramethylammonium. The bimolecular rate con stant for reaction with diisopropyl fluorophosphate was reduced 10 000-fold , indicating severe alteration in the binding area in W430A. The k(cat) val ues for butyrylthiocholine, o-nitrophenyl butyrate, and succinyldithiocholi ne were lower. This suggested that the mutation had caused misfolding of th e active site gorge without altering the Omega loop conformation/dynamics. W430 as well as W231 and W82 arrear to form the wall of the active site gor ge. Mutation of any of these tryptophans disrupts the architecture of the a ctive site. (C) 2001 Elsevier Science B.V. All rights reserved.