No abstract

Molecular modeling (GEMM 7.3) and molecular mechanics calculations (YET1 V 5.3) using the X-ray coordinates for acetylcholinesterase (AChE) from Torpedo culifornica indicate electrostatic stabilization by the active site, Glu-199, of the developing positive charge on the incipient carbonium ion in the dealkylation in the adducts of AChE with PsC, and P&s diastereomers of 2-(3,3dimethylbutyl) methylphosphonofluoridate (soman). His-440 is indispensable as a general acid catalyst of Ca bond breaking in the dealkylation reaction and that of bond breaking to the Ser r-0 in reactivation. This demand for catalysis seems to be satisfied for the reactivation of enzyme from the PsC, diastereomer of soman, but not from the P(S)C(R) diastereomer.Acetylcholinesterase inhibition; Serine hydrolase inhibition; Irreversible enzyme inhibition; Enzyme inhibition by organophosphorus compound

Huperzine A, a potential agent for therapy in Alzheimer's disease and for prophylaxis of organophosphate toxicity, has recently been characterized as a reversible inhibitor of cholinesterases. To examine the specificity of this novel compound in more detail, we have examined the interaction of the 2 stereoisomers of Huperzine A with cholinesterases and site-specific mutants that detail the involvement of specific amino acid residues. Inhibition of fetal bovine serum acetylcholinesterase by (-)-Huperzine A was 35-fold more potent than (+)-Huperzine A, with K , values of 6.2 nM and 210 nM, respectively. In addition, (-)-Huperzine A was 88-fold more potent in inhibiting Torpedo acetylcholinesterase than (+)-Huperzine A, with K , values of 0.25 pM and 22 pM, respectively. Far larger K , values that did not differ between the 2 stereoisomers were observed with horse and human serum butyrylcholinesterases. Mammalian acetylcholinesterase, Torpedo acetylcholinesterase, and mammalian butyrylcholinesterase can be distinguished by the amino acid Tyr, Phe, or Ala in the 330 position, respectively. Studies with mouse acetylcholinesterase mutants, Tyr 337(330) Phe and Tyr 337(330) Ala yielded a difference in reactivity that closely mimicked the native enzymes. In contrast, mutation of the conserved Glu 199 residue to Gln in Torpedo acetylcholinesterase produced only a 3-fold increase in K , value for the binding of Huperzine A. Molecular mechanics energy minimization of the complexes formed between each of the 2 stereoisomers of Huperzine A and fetal bovine serum acetylcholinesterase, Torpedo acetylcholinesterase, or human butyrylcholinesterase also revealed that (-)-Huperzine A gave a better fit than (+)-Huperzine A and implicated Tyr 337(330) in the stereoselectivity of Huperzine A.