Abstract
Cholinesterases (ChE’s) are readily phosphylated at the active site serine, by a variety of organophosphorus agents (OP) (Aldrich et al., 1972; Taylor, 1990). The enzyme can be reactivated by various oxime nucleophiles but in certain cases reactivation is thwarted due to a concomitant unimolecular process termed aging (Aldrich et al., 1972). The aged enzyme-OP-conjugate is refractive to reactivation (Gray, 1984) and thus renders treatment, following intoxication with certain OP insecticides or nerve gas agents, extremely difficult (Glickman et al., 1984). Recombinant human acetylcholinesterase (HuAChE) and several selected active site gorge mutants: W86A, E202Q, F295A, F297A, Y337A and E450A of HuAChE were studied with respect to catalytic activity towards charged and noncharged substrates, phosphylation by organophosphorus (OP) inhibitors: DFP, paraoxon and soman and subsequent aging of the resulting OP-conjugates. On the basis of these studies we have identified some of the critical elements in the active center that determine specificity to various OP-agents. Trp86, a key element of the active center “anionic subsite” that stabilizes noncovalent complexes of charged ligands by cation - π interactions (Ordentlich et al., 1993a) also contributes to chemical reactivity towards various noncharged OP - inhibitors as manifested by a 5–20 fold decrease in rates of phosphorylation observed for W86A mutant. Replacement of Tyr337 by alanine had a minimal effect on the bimolecular rate constant of inhibition by the OP-molecules. The 20-fold increase in phosphorylation rates of the F295A mutated enzyme by paraoxon is attributed to the better accommodation of the ethoxy moiety of the inhibitor by the less bulky alanine residue. This is consistent with the role of Phe295 in conferring specificity to the acyl pocket (Ordentlich et al., 1993a). The reduction in catalytic efficiency displayed by the E202Q and E450A mutants, and the marked decrease in the rates of both phosphylation and aging is consistent with the proposed role of these residues as key elements of the hydrogen bond network (Ordentlich et al., 1993b). The role of this network is to maintain the functional architecture of the active center and proper positioning of E202, thereby stabilizing the evolving transition states of acylation and phosphylation. The impaired “aging” process displayed by the E202Q and E450A mutants provides a basis for the engineering of novel reactivatable bioscavengers.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Aldrich, W.N., and Reiner, E. (1972). Enzyme Inhibitors as Substrates, North-Holland Publishing Co., Amsterdam.
Glickman, A.H., Wing, K.D. and Casida, J.E. (1984). Toxicol. Appl. Pharmacol. 73, 16–22.
Gray, A.P. (1984). Drug Metabolism Rev. 15, 557–589.
Ordentlich, A., Barak, D., Kroman, C., Flashner, Y., Leitner, M., Segall, Y., Ariel, N., Cohen, S., Velan, B. and Shafferman, A. (1993a). J. Biol. Chem. 268, 17083–17095.
Ordentlich, A., Kronman, C., Barak, D., Stein, D., Ariel, N., Marcus, D., Velan, B. and Shafferman, A. (1993b). FEBS Lett. 334, 215–220.
Taylor, P. (1990), in: The Pharmacological Basis of Therapeutics, 8th Ed. (Gilman, A.G., Rall, T.W., Nies, A.S. and Taylor, P., Eds.), pp 131–149, Macmillan Publishing Co., New York.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1995 Springer Science+Business Media New York
About this chapter
Cite this chapter
Ordentlich, A. et al. (1995). Amino Acids Determining Specificity to OP-Agents and Facilitating the “Aging” Process in Human Acetylcholinesterase. In: Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P. (eds) Enzymes of the Cholinesterase Family. Springer, Boston, MA. https://doi.org/10.1007/978-1-4899-1051-6_43
Download citation
DOI: https://doi.org/10.1007/978-1-4899-1051-6_43
Publisher Name: Springer, Boston, MA
Print ISBN: 978-1-4899-1053-0
Online ISBN: 978-1-4899-1051-6
eBook Packages: Springer Book Archive