SELECTIVE INACTIVATION OF BUTYRYLCHOLINESTERASE WITH METAL CHELATORS SUGGESTS THERE IS MORE THAN ONE METAL-BINDING SITE

Cholinesterases exhibit functions apart from their esterase activity, We have demonstrated an aryl acylamidase and a zinc stimulated metallo carboxypeptidase activity in human serum butyrylcholinesterase. To est ablish the presence of zinc binding sites in the enzyme we examined th e effect of metal chelators on its catalytic activities. The metal che lators 1,10-phenanthroline and N,N,N',N'-tetrakis (2-pyridyl methyl)et hylene diamine (TPEN) inhibited all the three catalytic activities in the enzyme. However, EDTA inhibited the peptidase activity exclusively without affecting the cholinesterase and aryl acylamidase activities. The catalytic activities were recovered upon removal of the chelator by Sephadex G-25 chromatography. Pre-treatment of the enzyme with any one of the three chelators resulted in the binding of the enzyme to a zinc-Sepharose column or to Zn-65(2+), Histidine modification of the e nzyme pretreated with chelators resulted in abolition of Zn-65(2+) bin ding and zinc-Sepharose binding. Whereas the binding studies demonstra ted removal of a metal from a Zn2+ binding site, attempts to remove th e metal responsible for catalytic activity were unsuccessful. Atomic a bsorption spectroscopy indicated approximately 2.5 mol of zinc per mol of enzyme before treatment with EDTA and 1 mol zinc per mol enzyme af ter EDTA treatment. The results indicate that there are at least two m etal binding sites on butyrylcholinesterase. The presence of two HXXE. ..H sequences in butyrylcholinesterase supports these findings. Our st udies implicate a zinc dependent metallocarboxypeptidase activity in n on-cholinergic functions of butyrylcholinesterase. (C) 1998 Elsevier S cience Ltd. All rights reserved.