Identification of amino acid residues involved in the binding of Huperzine A to cholinesterases

• Published in: Protein science Vol. 3; no. 10; pp. 1770 - 1778
• Main Authors: Saxena, Ashima, Doctor, Bhupendra P., Qian, Naifeng, Kovach, Ildiko M., Kozikowski, A.P., Pang, Y.P., Vellom, Daniel C., Radic, Zoran, Quinn, Daniel, Taylor, Palmer
• Format: Journal Article
• Language: English
• Published: Bristol Cold Spring Harbor Laboratory Press 01.10.1994
• Subjects: Acetylcholinesterase - chemistry; Acetylcholinesterase - metabolism; Alkaloids; Amino Acids - analysis; Amino Acids - metabolism; Animals; Binding Sites; Butyrylcholinesterase - chemistry; Butyrylcholinesterase - metabolism; Cholinesterase Inhibitors - metabolism; Cholinesterase Inhibitors - pharmacology; cholinesterases; Cholinesterases - chemistry; Cholinesterases - metabolism; Electrochemistry; Humans; Huperzine A; Hydrogen Bonding; inhibitor; Kinetics; Mice; Models, Molecular; molecular modeling; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Sesquiterpenes - chemistry; Sesquiterpenes - metabolism; Sesquiterpenes - pharmacology; site‐directed mutagenesis; Stereoisomerism; Thermodynamics; Torpedo
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1002/pro.5560031017

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 K1 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 K1 values of 0.25 μM and 22 μM, respectively. Far larger K1 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 K1 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.