Purification and properties of valyl-tRNA synthetase from Mycobacterium smegmatis

Abstract

Valyn-tRNA synthetase from Mycobacterium smegmatis has been purified over 1200-fold by conventional techniques as well as affinity chromatography on valyl-aminohexyl Sepharose columns. The purified preparation is homogeneous by electrophoretic and immunologic criteria. The enzyme is a tetramer of approximate molecular weight of 120 000, composed of a single type of subunit. The synthetase exhibited maximal activity between 35-40°C and pH 6.8-7.0. The pure enzyme though stable for several months below 0°C, loses activity completely at 70°C, for 1 min. The enzyme showed normal Michaelis-Menten kinetic behaviour in the total aminoacylation reaction with values of 1.25 μM, 0.1 mM and 1.0 μM for valine, ATP and tRNA, respectively, but the kinetic response deviated from the above pattern in the partial (activation) reaction. Based on these findings, the existence of the enzyme in two molecular forms, modulated by substrate concentration has been suggested; of these, only one may be active in the total reaction, while both forms may function in the prophosphate exchange reaction.