Arginyl-tRNA synthetase from Escherichia coli, purification by affinity chromatography, properties, and steady-state kinetics.
A Blue Sephadex G-150 affinity column adsorbs the arginyl-tRNA synthetase of Escherichia coli K12 and purifies it with high efficiency. The relatively low enzyme content was conveniently purified by DEAE-cellulose chromatography, affinity chromatography, and fast protein liquid chromatography to a preparation with high activity capable of catalyzing the esterification of about 23,000 nmol of arginine to the cognate tRNA per milligram of enzyme within 1 min, at 37 degrees C, pH 7.4. The turnover number is about 27 s-1. The purification was about 1200-fold, and the overall yield was more than 30%. The enzyme has a single polypeptide chain of about Mr 70,000 and binds arginine and tRNA with 1:1 stoichiometry. For the aminoacylation reaction, the Km values at pH 7.4, 37 degrees C, for various substrates were determined: 12 microM, 0.9 mM, and 2.5 microM for arginine, ATP, and tRNA, respectively. The Km value for cognate tRNA is higher than those of most of the aminoacyl-tRNA synthetase systems so far reported. The ATP-PPi exchange reaction proceeds only in the presence of arginine-specific tRNA. The Km values of the exchange at pH 7.2, 37 degrees C, are 0.11 mM, 2.9 mM, and 0.5 mM for arginine, ATP, and PPi, respectively, with a turnover number of 40 s-1. The pH dependence shows that the reaction is favored toward slightly acidic conditions where the aminoacylation is relatively depressed.
Lin
S X
SX
Shanghai Institute of Biochemistry, Academia Sinica, China.
Shi
J P
JP
Cheng
X D
XD
Wang
Y L
YL
D000604:Amino Acyl-tRNA Synthetases / Q000302:isolation & purification*; D001126:Arginine-tRNA Ligase / Q000302:isolation & purification* / Q000378:metabolism; D001665:Binding Sites; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight
D000604:Amino Acyl-tRNA Synthetases; D001126:Arginine-tRNA Ligase