Purification of isoleucyl‐tRNA synthetase from Methanobacterium thermoautotrophicum by pseudomonic acid affinity chromatography

Abstract

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°C. Pseudomonic acid is a strong competitive inhibitor of the aminoacylation reaction with respect to both l‐isoleucine (KIlei 10 nM) and ATP (KATPi 20 nM). Copyright © 1989, Wiley Blackwell. All rights reserved