Thiol Ligation of Two Zinc Atoms to a Class I tRNA Synthetase: Evidence for Unshared Thiols and Role in Amino Acid Binding and Utilization

• Published in: Biochemistry (Easton) 1994-11, Vol.33 (47), p.14213-14220
• Main Authors: Landro, James A, Schmidt, Eric, Schimmel, Paul, Tierney, David L, Penner-Hahn, James E
• Format: Article
• Language: English
• Subjects: Amino Acids - metabolism; Binding Sites; Cobalt; Cysteine - metabolism; Escherichia coli; Isoleucine - metabolism; Isoleucine-tRNA Ligase - chemistry; Isoleucine-tRNA Ligase - genetics; Isoleucine-tRNA Ligase - metabolism; Kinetics; Mutagenesis, Site-Directed; Spectrophotometry, Ultraviolet; Spectrum Analysis; Structure-Activity Relationship; Sulfhydryl Compounds - metabolism; X-Rays; Zinc - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00251a033

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- and isoleucyl-tRNA synthetases each have a single zinc atom coordinated to ligands contained in the catalytic domain. Isoleucyl-tRNA synthetase has a second, functionally essential, zinc bound to ligands at the C-terminal end of the 939 amino acid polypeptide. Recent evidence suggested that this structure curls back and interacts directly or indirectly with the active site. We show here by X-ray absorption spectroscopy that the average Zn environment contains predominantly sulfur ligands with a Zn-S distance of 2.33 A. A model with eight coordinated thiolates divided between two Zn(Cys)4 structures best fit the data which are not consistent with a thiolate-bridged Zn2(Cys)6 structure 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 important 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 incorporated into the class-defining catalytic domain common to class I enzymes, the architecture of at least some parts of the amino acid binding sites may differ from enzyme to enzyme and include motifs that bind zinc.