European Journal of Biochemistry, Vol 128, 41-46, Copyright © 1982 by Federation of European Biochemical Societies


Removal of the tightly bound zinc from Escherichia coli trypsin- modified methionyl-tRNA synthetase

JF Mayaux, T Kalogerakos, KK Brito and S Blanquet

The study of the behaviour of Escherichia coli methionyl-tRNA synthetase with chelating agents has shown that only 1,10- phenanthroline has an inhibitory effect on the tRNAMet aminoacylation activity. Under identical buffer conditions the isotopic [32P]PPi-ATP exchange activity is insensitive. Dialysis of the enzyme against 1,10- phenanthroline causes a slow loss of zinc from the enzyme which is paralleled by an irreversible loss of both the aminoacylation and isotopic exchange activities. The loss of zinc becomes faster upon the addition of small amounts of guanidine hydrochloride to the dialysis buffer containing phenanthroline, presumably by partially unfolding the protein. Studies of the reversible denaturation of the enzyme by 5 M guanidine hydrochloride shows that the inclusion of EDTA produces an enzyme species that has lost both zinc and activity. The inactive apoenzyme prepared in guanidine and EDTA can regain activity by dilution in a zinc-containing buffer.