Probing the metal binding sites of Escherichia coli isoleucyl-tRNA synthetase.
The metal binding properties of isoleucyl-tRNA synthetase (IleRS) from Escherichia coli were studied by in vivo substitution of the enzyme-bound metals. Purified E. coli IleRS was shown to have two tightly bound zinc atoms per active site. Cobalt- and cadmium-substituted IleRS were also found to contain two tightly bound Co2+ and Cd2+ atoms per polypeptide chain, respectively. The d-d transitions in the low energy absorption spectrum of Co(2+)-substituted IleRS were characteristic of that expected for two tetrahedrally coordinated Co2+ metals. Apo-IleRS was found to be inactive in both the aminoacylation of tRNA(Ile) and in the isoleucine-dependent ATP-pyrophosphate exchange reactions. Both Co(2+)- and Cd(2+)-substituted IleRS were found to have kcat/Km values in the isoleucine-dependent ATP-pyrophosphate exchange assay approximately 5-fold lower than the native Zn2+ enzyme. A single enzyme-bound Zn2+ or Co2+ atom per polypeptide chain could be removed by dialysis of Zn(2+)- or Co(2+)-substituted IleRS against 1,10-phenanthroline. Removal of one of the two enzyme-bound Zn2+ atoms per polypeptide chain with 1,10-phenanthroline was found to decrease (kcat/Km)Ile by approximately 130-fold. The dependence of the kinetic parameters on the identity and number of enzyme-bound metals in the isoleucine-dependent ATP-pyrophosphate exchange reaction suggests that at least one enzyme-bound metal is indirectly involved in aminoacyladenylate formation. Metal substitution or removal of one of the two enzyme-bound metals in IleRS was found to have little effect on the Km value for tRNA(Ile) or the kcat value for aminoacylation of tRNA(Ile).(ABSTRACT TRUNCATED AT 250 WORDS)
Xu
B
B
Department of Biochemistry and Molecular Biology, University of Texas Medical School-Houston, Medical School 77225.
Trawick
B
B
Krudy
G A
GA
Phillips
R M
RM
Zhou
L
L
Rosevear
P R
PR
D000215:Acylation; D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D002104:Cadmium / Q000378:metabolism; D003035:Cobalt / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007533:Isoleucine-tRNA Ligase / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D010618:Phenanthrolines / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D012355:RNA, Transfer, Ile / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D015032:Zinc / Q000378:metabolism
D010618:Phenanthrolines; D010710:Phosphates; D012355:RNA, Transfer, Ile; D002104:Cadmium; D003035:Cobalt; D000255:Adenosine Triphosphate; D007533:Isoleucine-tRNA Ligase; D015032:Zinc; C025205:1,10-phenanthroline