Evidence for distinct locations for metal binding sites in two closely related class I tRNA synthetases.
Of the ten class I tRNA synthetases, those for methionine and isoleucine are among the most closely related. In recent work we showed that the 676 amino acid E. coli methionine tRNA synthetase has one zinc bound per polypeptide. Zinc may be replaced by spectroscopically observable cobalt with retention of full activity. Bound zinc has been localized to a cysteine cluster within an insertion into the nucleotide binding fold that characterizes all class I enzymes. Mutations which interfere with metal ligation to these cysteines yield proteins that are defective in activity. Additional data presented here show that change of the cobalt oxidation state and coordination geometry of the Co(II)-substituted enzyme results in a complete loss in activity, and that mutations which replace any one of the zinc-binding cysteine sulfhydryls have a small but measurable effect on protein stability. These results further support the importance of the metal for the active site. We also show that, in contrast to methionine tRNA synthetase, the closely related but larger 939 amino acid E. coli isoleucine tRNA synthetase contains 1.5 to 2 molecules of zinc bound per polypeptide. The cobalt-substituted enzyme is active and shows the expected spectrum for tetrahedral coordination to sulfur ligands. Although the site(s) for metal coordination in isoleucine tRNA synthetase has not been rigorously established, one likely sequence element is in a region of the primary structure 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.
Schimmel
P
P
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Landro
J A
JA
Schmidt
E
E
D000595:Amino Acid Sequence; D001665:Binding Sites; D003035:Cobalt / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D007533:Isoleucine-tRNA Ligase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008718:Methionine-tRNA Ligase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary; D013054:Spectrophotometry, Atomic; D013696:Temperature; D015032:Zinc / Q000378:metabolism*
D003035:Cobalt; D008718:Methionine-tRNA Ligase; D007533:Isoleucine-tRNA Ligase; D015032:Zinc