THIOL LIGATION OF 2 ZINC ATOMS TO A CLASS-I TRANSFER-RNA SYNTHETASE -EVIDENCE FOR UNSHARED THIOLS AND ROLE IN AMINO-ACID BINDING AND UTILIZATION

Class I tRNA synthetases generally contain a characteristic N-terminal catalytic core joined to a C-terminal domain that is idiosyncratic to the enzyme. The closely related class I Escherichia coli methionyl- a nd isoleucyl-tRNA synthetases each have a single zinc atom coordinated to ligands contained in the catalytic domain. Isoleucyl-tRNA syntheta se has a second, functionally essential, zinc bound to ligands at the C-terminal end of the 939 amino acid polypeptide. Recent evidence sugg ested that this structure curls back and interacts directly or indirec tly with the active site. We show here by X-ray absorption spectroscop y that the average Zn environment contains predominantly sulfur ligand s with a Zn-S distance of 2.33 Angstrom. A model with eight coordinate d thiolates divided between two Zn(Cys)(4) structures best fit the dat a which are not consistent with a thiolate-bridged Zn-2(Cys)(6) struct ure joining the C-terminal end with the N-terminal active site domain. We also show that zinc bound to the N-terminal catalytic core is impo rtant specifically for amino acid binding and utilization, although a direct interaction with zinc is unlikely. We suggest that, in addition to idiosyncratic sequences for tRNA acceptor helix interactions incor porated into the class-defining catalytic domain common to class I enz ymes, the architecture of at least some parts of the amino acid bindin g sites may differ from enzyme to enzyme and include motifs that bind zinc.