Removal of the Tightly Bound Zinc from Escherichia coli Trypsin-Modified Methionyl-tRNA Synthetase

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Abstract
Study of the behavior of E. 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 Zn from the enzyme which is paralleled by an irreversible loss of both the aminoacylation and isotopic exchange activities. The loss of Zn 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 show that the inclusion of EDTA produces an enzyme species that has lost both Zn and activity. The inactive apoenzyme prepared in guanidine and EDTA can regain activity by dilution in a Zn-containing buffer.