EVIDENCE FOR DISTINCT LOCATIONS FOR METAL-BINDING SITES IN 2 CLOSELY-RELATED CLASS-I TRANSFER-RNA SYNTHETASES

Of the ten class I tRNA synthetases, those for methionine and isoleuci ne are among the most closely related. In recent work we showed that t he 676 amino acid E. coli methionine tRNA synthetase has one zinc boun d per polypeptide. Zinc may be replaced by spectroscopically observabl e cob alt with retention of full activity. Bound zinc has been localiz ed to a cysteine cluster within an insertion into the nucleotide bindi ng fold that characterizes all class I enzymes. Mutations which interf ere with metal ligation to these cysteines yield proteins that are def ective in activity. Additional data presented here show that change of the cobalt oxidation state and coordination geometry of the Co(II)-su bstituted enzyme results in a complete loss in activity, and that muta tions which replace any one of the zinc-binding cysteine sulfhydryls h ave a small but measurable effect on protein stability. These results further support the importance of the metal for the active site. We al so show that, in contrast to methionine tRNA synthetase, the closely r elated but larger 939 amino acid E. coli isoleucine tRNA synthetase co ntains 1.5 to 2 molecules of zinc bound per polypeptide. The cob alt-s ubstituted enzyme is active and shows the expected spectrum for tetrah edral coordination to sulfur ligands. Although the site(s) for metal c oordination in isoleucine tRNA synthetase has not been rigorously esta blished, one likely sequence element is in a region of the primary str ucture different from the known metal binding site in methionine tRNA synthetase. Thus, these two closely related proteins have incorporated metal binding sites into distinct parts of their related sequences.