EVIDENCE FOR A ZN2-BINDING SITE IN HUMAN SERUM BUTYRYLCHOLINESTERASE()

Purified human serum butyrylcholinesterase after treatment with either of the metal chelators EDTA or NaCN was able to bind to a Zn2+-chelat e-Sepharose affinity column and was eluted from the column by EDTA or imidazole. Prior EDTA treatment of the enzyme was essential for bindin g to this affinity column. The enzyme could be labelled with Zn-65(2+) after EDTA treatment of the enzyme. Diethylpyrocarbonate modification of histidine residues in the EDTA-treated enzyme resulted in the abol ition of both binding to the Zn2+-chelate-Sepharose column and labelli ng by Zn-65(2+). Stoicheiometry of Zn-65(2+) binding indicated approxi mately 0.85 mol of Zn2+/mol of subunit of the EDTA-treated enzyme. EDT A or NaCN treatment resulted in the loss of thermal stability of the e nzyme at 37 degrees C which could not be reversed by Zn2+. Whereas the cholinesterase activity of butyryl cholinesterase was not affected by EDTA, there was significant loss of its carboxypeptidase activity in the presence of EDTA, and the loss could be reversed by added ZnCl2. T hese results suggest the presence of a Zn2+-binding site on human seru m butyrylcholinesterase and the involvement of histidine residues in t he metal binding. The presence in human serum butyrylcholinesterase of a sequence HXXE...H found in many known Zn2+-containing enzymes suppo rts these findings.