Studies on aminoacyl-TRNA synthestases from mycobacteria

Abstract

As a part of the investigations on “Genetic coding and protein synthesis in Mycobacteria”, studies on aminoacyl?tRNA synthetases have been undertaken.

Valyl?tRNA synthetase from M. smegmatis has been purified by conventional techniques as well as affinity chromatography on valyl?aminohexyl Sepharose. The purified enzyme is homogeneous electrophoretically and antigenically. It is a tetramer of approximate molecular weight 122,000 composed of a single type of subunit. The synthetase shows normal Michaelis–Menten kinetic behaviour in the aminoacylation reaction but deviates from that pattern in the partial 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 pyrophosphate?exchange reaction. For comparison of kinetic properties, the isoleucyl?tRNA synthetase from M. smegmatis has also been purified to homogeneity. This enzyme shows normal Michaelis–Menten kinetics in both total and partial reactions.

Valyl?tRNA synthetase from the slow?growing, human pathogenic strain M. tuberculosis has also been purified to homogeneity. This enzyme is a monomer of approximate molecular weight 98,000 and shows Michaelis–Menten kinetics in both partial and total reactions. The properties of this enzyme have been compared with those of the enzyme from M. smegmatis.

Attempts have also been made to characterize the mechanism of the synthetase reaction using the influence of cations (Mg²? and polyamines) on the pure enzymes from M. smegmatis. Polyamines alone, in the complete absence of divalent metal ions, do not satisfy the cationic requirement in the enzymatic aminoacylation of tRNA. However, they stimulate the aminoacylation reaction in the presence of suboptimal concentrations of Mg²?. The inhibitory effects of high concentrations of KCl and high pH on aminoacylation of tRNA are also counteracted by spermine. Detailed kinetic analysis of the polyamine effects suggests that at optimal Mg²? concentrations, aminoacylation follows the ping?pong mechanism; however, at low Mg²? concentrations and in the presence of spermine, the reaction appears to proceed by the concerted mechanism.

The effect of ethidium bromide on aminoacylation of tRNA has also been studied. The inhibitory effect of ethidium bromide is prevented by increasing concentrations of Mg²? or, more efficiently, by spermidine. Dixon?plot analysis of the data shows that ethidium bromide is a competitive inhibitor of both Mg²? and spermidine in the aminoacylation reaction.