Studies on Arginyl- and lysyl- tRNA synthetases from mycobcterium smegmatis

Abstract

STUDIES ON ARGINYL- AND LYSYL-tRNA SYNTHETASES FROM MYCOBACTERIUM SMEGMATIS

Aminoacyl-tRNA synthetases are a family of enzymes which play an important role in protein synthesis. These enzymes catalyze the esterification of amino acids to tRNA with the accompanying hydrolysis of ATP to AMP and PPi. Aminoacyl-tRNA synthetases have been purified from various sources and studied for their structural and enzymatic properties. Amongst prokaryotic sources, however, the investigations have mostly been confined to E. coli and Bacillus species. Some controversy has centered on the question of whether the reaction proceeds via a one-step sequential or two-step ping-pong mechanism.

Arginyl-tRNA synthetase, along with glutamyl- and glutaminyl-tRNA synthetases, is of special interest because, in contrast to the seventeen other aminoacyl-tRNA synthetases, it catalyzes the ATP–PPi exchange only in the presence of cognate tRNA.

Arginyl-tRNA synthetase and lysyl-tRNA synthetase from Mycobacterium smegmatis SN2 have been purified to an electrophoretically homogeneous state. The purification protocol consisted of ion-exchange and affinity chromatographic techniques. Arginyl-tRNA synthetase was purified over 1650-fold with a yield of 14%, while lysyl-tRNA synthetase was purified over 686-fold with a yield of 7%. The molecular activities of arginyl- and lysyl-tRNA synthetases were 0.37 min?¹ and 0.35 min?¹, respectively. Arginyl-tRNA synthetase was found to be a monomer of M = 56,000. Lysyl-tRNA synthetase was a dimer of M = 126,000 and was composed of identical subunits. The subunit structure and molecular weight of both enzymes resemble those isolated from other sources. The optimal pH, temperature, and Mg²? concentrations for aminoacylation and the K? values for the three substrates have been determined.

Initial velocity studies for three-substrate enzymes allow segregation of kinetic mechanisms into sequential and ping-pong classes, and in addition often define the specific mechanism. A detailed analysis of the kinetic mechanism of arginyl- and lysyl-tRNA synthetases from M. smegmatis has been carried out as described by Cleland as well as Fromm. The results of the initial velocity and product inhibition studies are consistent with a rapid-equilibrium random ternary mechanism for both arginyl- and lysyl-tRNA synthetases from M. smegmatis. Polyamines stimulated the formation of arginyl-tRNA in the presence of sub-optimal Mg²? concentrations. The kinetic analysis was performed under two reaction conditions: (i) in the presence of sub-optimal Mg²? and spermine, and (ii) optimal Mg²?. The kinetic patterns obtained indicated that the mechanism remained random sequential under both reaction conditions.

In a complex reaction of the kind catalyzed by aminoacyl-tRNA synthetases, in which three substrates participate and three products are formed, partial reactions can be investigated experimentally. The different attempts to reveal formation of enzyme-bound aminoacyl-adenylate provided no evidence for its existence. While tRNA was absolutely essential for the ATP–PPi exchange in the case of arginyl-tRNA synthetase, it stimulated the exchange catalyzed by lysyl-tRNA synthetase. Both arginyl- and lysyl-tRNA synthetases were unable to utilize synthetic aminoacyl-adenylate as a substrate in the pyrophosphorolysis reaction even in the presence of tRNA. The reverse reaction, studied by the deacylation of aminoacyl-tRNA, was dependent on the presence of both AMP and PPi. Thus, like the forward reaction, the reverse reaction also requires four components in a quaternary complex: E·aminoacyl-tRNA·AMP·PPi for the reaction to occur.

Arginyl- and lysyl-tRNA synthetases belong to two different classes of aminoacyl-tRNA synthetases, the former requiring cognate tRNA to catalyze the ATP–PPi exchange reaction. Yet, the detailed kinetic analyses of both these enzymes from M. smegmatis have revealed a common kinetic mechanism under the reaction conditions used.