Acetylcholinesterase active centre and gorge conformations analysed by combinatorial mutations and enantiomeric phosphonates

Abstract

A series of eight double and triple mutants of mouse acetylcholinesterase (AChE; EC 3.1.1.7), with substitutions corresponding to residues found largely within the butyrylcholinesterase (BChE; EC 3.1.1.8) active-centre gorge, was analysed to compare steady-state kinetic constants for substrate turnover and inhibition parameters for enantiomeric methylphosphonate esters. The mutations combined substitutions in the acyl pocket (Phe295→Leu and Phe297→Ile) with the choline-binding site (Tyr337→Ala and Phe338→Ala) and with a side chain (Glu202→ Gln) N-terminal to the active-site serine, Ser203. The mutations affected catalysis by increasing Km and decreasing kcat, but these constants were typically affected by an order of magnitude or less, a relatively small change compared with the catalytic potential of AChE. To analyse the constraints on stereoselective phosphonylation, the mutant enzymes were reacted with a congeneric series of SP- and RP-methylphosphonates of known absolute stereochemistry. Where possible, the overall reaction rates were deconstructed into the primary constants for formation of the reversible complex and intrinsic phosphonylation. The multiple mutations greatly reduced the reaction rates of the more reactive SP-methylphosphonates, whereas the rates of reaction with the RP-methylphosphonates were markedly enhanced. With the phosphonates of larger steric bulk, the enhancement of rates for the RP enantiomers, coupled with the reduction of the SP enantiomers, was sufficient to invert markedly the enantiomeric preference. The sequence of mutations to enlarge the size of the AChE active-centre gorge, resembling in part the more spacious gorge of BChE, did not show an ordered conversion into BChE reactivity as anticipated for a rigid template. Rather, the individual aromatic residues may mutually interact to confer a distinctive stereospecificity pattern towards organophosphates.