Purification of isoleucyl-tRNA synthetase from Methanobacterium thermoautotrophicum by pseudomonic acid affinity chromatography.
The isoleucyl-tRNA synthetase of the archaebacterium Methanobacterium thermoautotrophicum was purified 1500-fold to electrophoretic homogeneity by a procedure based on affinity chromatography on Sepharose-bound pseudomonic acid, a strong competitive inhibitor of this enzyme. The purified enzyme is a monomer with a molecular mass of 120 kDa. In this respect and in its Km values for the PPi-ATP exchange, and aminoacylation reactions, it resembles the isoleucyl-tRNA synthetases from eubacterial and eukaryotic sources. Its aminoacylation activity is optimal at pH 8.0 and at 55 degrees C. Pseudomonic acid is a strong competitive inhibitor of the aminoacylation reaction with respect to both L-isoleucine (KiIle 10 nM) and ATP (KiATP 20 nM).
Rechsteiner
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Mikrobiologisches Institut, Eidgenössische Technische Hochschule, ETH-Zentrum, Zürich, Switzerland.
Leisinger
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D000604:Amino Acyl-tRNA Synthetases / Q000302:isolation & purification*; D000900:Anti-Bacterial Agents; D002846:Chromatography, Affinity / Q000379:methods; D019605:Euryarchaeota / Q000201:enzymology*; D005227:Fatty Acids; D007533:Isoleucine-tRNA Ligase / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D016712:Mupirocin
D000900:Anti-Bacterial Agents; D005227:Fatty Acids; D016712:Mupirocin; D000604:Amino Acyl-tRNA Synthetases; D007533:Isoleucine-tRNA Ligase