Mechanistic Studies of Reaction Coupling in Glu-tRNAGln Amidotransferase

Abstract

Organisms lacking Gln-tRNA synthetase produce Gln-tRNA^Gln from misacylated Glu-tRNA^Gln through the transamidation activity of Glu-tRNA^Gln amidotransferase (Glu-AdT). Glu-AdT hydrolyzes Gln to Glu and NH₃, using the latter product to transamidate Glu-tRNA^Gln in concert with ATP hydrolysis. In the absence of the amido acceptor, Glu-tRNA^Gln, the enzyme has basal glutaminase activity that is unaffected by ATP. However, Glu-tRNA^Gln activates the glutaminase activity of the enzyme about 10-fold; addition of ATP elicits a further 7-fold increase. These enhanced activities mainly result from increases in k_cat without significant effects on the K_m for Gln. To determine if ATP binding is sufficient to induce full activation, we tested a variety of ATP analogues for their ability to stimulate tRNA-dependent glutaminase activity. Despite their binding to Glu-AdT, none of the ATP analogues induced glutaminase activation except ATP-γS, which stimulates glutaminase activity to the same level as ATP, but without formation of Gln-tRNA^Gln. ATP-γS hydrolysis by Glu-AdT is very low in the absence or presence of Glu-tRNA^Gln and Gln. In contrast, Glu-tRNA^Gln stimulates basal ATP hydrolysis slightly, but full activation of ATP hydrolysis requires both Gln and Glu-tRNA^Gln. Simultaneous monitoring of ATP or ATP-γS hydrolysis and glutaminase and transamidase activities reveals tight coupling among these activities in the presence of ATP, with all three activities waning in concert when Glu-tRNA^Gln levels become exhausted. ATP-γS stimulates the glutaminase activity to an extent similar to that with ATP, but without concomitant transamidase activity and with a very low level of ATP-γS hydrolysis. This uncoupling between ATP-γS hydrolysis and glutaminase activities suggests that the activation of glutaminase activity by ATP or ATP-γS, together with Glu-tRNA^Gln, results either from an allosteric effect due simply to binding of these analogues to the enzyme or from some structural changes that attend ATP or ATP-γS hydrolysis.