Thiol ligation of two zinc atoms to a class I tRNA 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- 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.
Landro
J A
JA
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Schmidt
E
E
Schimmel
P
P
Tierney
D L
DL
Penner-Hahn
J E
JE
D000596:Amino Acids / Q000378:metabolism*; D001665:Binding Sites; D003035:Cobalt; D003545:Cysteine / Q000378:metabolism; D007532:Isoleucine / Q000378:metabolism; D007533:Isoleucine-tRNA Ligase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D013056:Spectrophotometry, Ultraviolet; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D014965:X-Rays; D015032:Zinc / Q000378:metabolism*
D000596:Amino Acids; D013438:Sulfhydryl Compounds; D007532:Isoleucine; D003035:Cobalt; D007533:Isoleucine-tRNA Ligase; D015032:Zinc; D003545:Cysteine