Identification of the metal ligands and characterization of a putative zinc finger in methionyl-tRNA synthetase.

A truncated form of the methionyl-tRNA synthetase (delta MTS), which has been cloned, overproduced, and characterized, was used in an attempt to better understand the role of the enzyme-bound zinc in the amino-acylation process. Apo-, Zn(2+)-, Co(2+)-, and 113Cd(2+)-substituted delta MTS proteins were prepared in vivo and purified to homogeneity. Apo-delta MTS was devoid of enzymatic activity in the aminoacylation of tRNA(fMet) and in the methionine-dependent ATP-pyrophosphate exchange reactions. Kinetic constants in both the aminoacylation and ATP-pyrophosphate exchange reactions for the Co(2+)- and 113Cd(2+)-substituted delta MTS proteins were found to be identical with those of the native Zn2+ protein. The low energy absorption spectrum of Co(2+)-substituted delta MTS resembles the d-d transition bands characteristic of tetrahedrally coordinated Co(2+)-substituted proteins. A strong S-->Co2+ charge transfer absorption at 350 nm was clearly evident having a molar absorptivity consistent with four thiolate ligands. The environment of the metal center was further probed by measuring the 113Cd chemical shift of 113Cd(2+)-substituted delta MTS. A single resonance at 759.6 ppm was observed. This chemical shift is consistent with Cd2+ coordinated to four thiolate ligands. The Escherichia coli methionyl-tRNA synthetase contains a potential metal binding sequence Cys-X2-Cys-X9-Cys-X2-Cys in a connecting polypeptide within the nucleotide fold. Titration of a 21-amino acid peptide corresponding to this putative metal binding site, Cys145-Cys161, was shown to bind Co2+ with a Kd of 120 +/- 11 microM. These results demonstrate that the isolated zinc finger binding domain is capable of specifically forming a stoichiometric complex with the divalent cation. Taken together, our studies identify the 4 cysteine residues in the zinc finger-like domain as the metal binding ligands in the E. coli methionyl-tRNA synthetase. The role of the enzyme-bound metal appears to be structural and not directly involved in catalysis.