ENGINEERING RESISTANCE TO AGING OF PHOSPHYLATED HUMAN ACETYLCHOLINESTERASE - ROLE OF HYDROGEN-BOND NETWORK IN THE ACTIVE-CENTER

Recombinant human acetylcholinesterase (HuAChE) and selected mutants (E202Q, Y337A, E450A) were studied with respect to catalytic activity towards charged and noncharged substrates, phosphylation with organophosphorus (OP) inhibitors and subsequent aging of the OP-conjugates. Amino acid E450, unlike residues E202 and Y337, is not within interaction distance from the active center. Yet, the bimolecular rates of catalysis and phosphylation are 30-100 fold lower for both E450A and E202Q compared to Y337A or the wild type and in both mutants the resulting OP-conjugates show striking resistance to aging. It is proposed that a hydrogen bond network, that maintains the functional architecture of the active center, involving water molecules and residues E202 and E450, is responsible for the observed behaviour.