Mechanistic studies of reaction coupling in Glu-tRNA(Gln) amidotransferase

Organisms lacking Gln-tRNA synthetase produce Gln- tRN(Gln) from misacylate d Glu-tRNA(Gln) through the transamidation activity of Glu-tRNA(Gln) amidot ransferase (Glu-AdT). Glu-AdT hydrolyzes Gin to Glu and NH3, using the latt er 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 glu taminase activity that is unaffected by ATP. However, Glu-tRNA(Gln) activat es the glutaminase activity of the enzyme about 10-fold; addition of ATP el icits a further 7-fold increase. These enhanced activities mainly result fr om increases in k(cat) without significant effects on the K-m for Gln. To d etermine if ATP binding is sufficient to induce full activation, we tested a variety of ATP analogues for their ability to stimulate tRNA-dependent gl utaminase activity. Despite their binding to Glu-AdT, none of the ATP analo gues induced glutaminase activation except ATP-gammaS, which stimulates glu taminase activity to the same level as ATP, but without formation of Gln-tR NA(Gln). ATP-gammaS hydrolysis by Glu-AdT is very low in the absence or pre sence of Glu-tRNA(Gln) and Gln. In contrast, Glu-tRNA(Gln) stimulates basal ATP hydrolysis slightly, but full activation of ATP hydrolysis requires bo th Gln and Glu-tRNA(Gln). Simultaneous monitoring of ATP or ATP-gammaS hydr olysis and glutaminase and transamidase activities reveals tight coupling a mong these activities in the presence of ATP, with all three activities wan ing in concert when Glu-tRNA(Gln) levels become exhausted. ATP-gammaS stimu lates the glutaminase activity to an extent similar to that with ATP, but w ithout concomitant transamidase activity and with a very low level of ATP-g ammaS hydrolysis. This uncoupling between ATP-gammaS hydrolysis and glutami nase activities suggests that the activation of glutaminase activity by ATP or ATP-gammaS, together with Glu-tRNA(Gln), results either from an alloste ric effect due simply to binding of these analogues to the enzyme or from s ome structural changes that attend ATP or ATP-gammaS hydrolysis.