Evidence for presence of Zn⁺²-binding site in acetylcholinesterase

Abstract

The purified electric eel acetylcholinesterase (AChE) was able to bind to the Zn⁺²-chelate-Sepharose affinity column only on treatment with EDTA. However goat brain and cobra venom AChE were binding to the column even without EDTA treatment. But all these enzymes were eluted from Zn⁺²-chelate-Sepharose affinity column by EDTA. Modification of histidine residues of AChEs by diethylpyrocarbonate resulted in abolition of its binding to Zn⁺²-chelate-Sepharose affinity column. Further the thermal stability of such EDTA-treated enzymes was decreased at 37 °C. Dot-blot studies involving ⁶⁵Zn⁺² further demonstrated the zinc binding property of AChE proteins. These results confirm the presence of Zn⁺²-binding site on AChE and further removal of metal from binding site with chelators resulted in loss of its catalytic function. Presence of metal-binding sequences HXXE...H in AChE, similar to many other metal containing enzymes supports our findings.