Structural Modifications of the Ω Loop in Human Acetylcholinesterase
The surface Ω loop (Cys69-Cys96) in human acetylcholinesterase (HuAChE) was recently implicated by molecular simulations in enhancing substrate and ligand accessibility to the active center and in the allosteric modulation of enzymatic activity. In lipases which together with AChEs belong to the esterase/lipase family sharing the α/β hydrolase fold, such Ω loop mobility was also shown to be functionally significant. In order to examine the relation between the loop structure and reactivity characteristics of the enzyme, several mutant HuAChE’s carrying modifications of the loop sequence were generated and kinetically evaluated. These modifications included replacements at the loop ends (Tyr72, Glu95), substitution of residues involved in hydrogen-bond interactions of the loop with the protein core (Tyr77, Glu84) as well as residues participating in such interactions within the loop (Met85, Asn87), replacement of the conserved proline residues (Pro 78, Pro 88) and deletion of 5 residue segment (Pro 78-Gly82) from the loop sequence. Most of the residue replacements, including those of the prolines, had very limited or no effect on enzyme reactivity. These findings suggest that unlike the case of lipase the Ω loop in HuAChE is not involved in in large lid-like motions. In cases where modifications of loop sequence had some effect on reactivity, the effects could be attributed to an altered position of residue Trp86 as implied by the altered catalytic activity, the interactions with quaternary ligands and the effect on the inhibitory activity of the peripheral site ligand propidium.