Mechanistic Studies of Reaction Coupling in Glu-tRNAGln Amidotransferase
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- 作者：Kurumi Y. Horiuchi ; Mark R. Harpel ; Li Shen ; Ying Luo ; Kelley C. Rogers ; and Robert A. Copeland
- 刊名：Biochemistry
- 出版年：2001
- 出版时间：May 29, 2001
- 年：2001
- 卷：40
- 期：21
- 页码：6450 - 6457
- 全文大小：72K
- 年卷期：v.40,no.21(May 29, 2001)
- ISSN：1520-4995
文摘
Organisms lacking Gln-tRNA synthetase produce Gln-tRNAGln from misacylated Glu-tRNAGlnthrough the transamidation activity of Glu-tRNAGln amidotransferase (Glu-AdT). Glu-AdT hydrolyzesGln to Glu and NH3, using the latter product to transamidate Glu-tRNAGln in concert with ATP hydrolysis.In the absence of the amido acceptor, Glu-tRNAGln, the enzyme has basal glutaminase activity that isunaffected by ATP. However, Glu-tRNAGln 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 increasesin kcat without significant effects on the Km for Gln. To determine if ATP binding is sufficient to inducefull activation, we tested a variety of ATP analogues for their ability to stimulate tRNA-dependentglutaminase activity. Despite their binding to Glu-AdT, none of the ATP analogues induced glutaminaseactivation except ATP-
S, which stimulates glutaminase activity to the same level as ATP, but withoutformation of Gln-tRNAGln. ATP-S hydrolysis by Glu-AdT is very low in the absence or presence ofGlu-tRNAGln and Gln. In contrast, Glu-tRNAGln stimulates basal ATP hydrolysis slightly, but full activationof ATP hydrolysis requires both Gln and Glu-tRNAGln. Simultaneous monitoring of ATP or ATP-Shydrolysis and glutaminase and transamidase activities reveals tight coupling among these activities inthe presence of ATP, with all three activities waning in concert when Glu-tRNAGln levels become exhausted.ATP-S stimulates the glutaminase activity to an extent similar to that with ATP, but without concomitanttransamidase activity and with a very low level of ATP-S hydrolysis. This uncoupling between ATP-Shydrolysis and glutaminase activities suggests that the activation of glutaminase activity by ATP or ATP-S, together with Glu-tRNAGln, results either from an allosteric effect due simply to binding of theseanalogues to the enzyme or from some structural changes that attend ATP or ATP-S hydrolysis.
S, which stimulates glutaminase activity to the same level as ATP, but withoutformation of Gln-tRNAGln. ATP-S hydrolysis by Glu-AdT is very low in the absence or presence ofGlu-tRNAGln and Gln. In contrast, Glu-tRNAGln stimulates basal ATP hydrolysis slightly, but full activationof ATP hydrolysis requires both Gln and Glu-tRNAGln. Simultaneous monitoring of ATP or ATP-Shydrolysis and glutaminase and transamidase activities reveals tight coupling among these activities inthe presence of ATP, with all three activities waning in concert when Glu-tRNAGln levels become exhausted.ATP-S stimulates the glutaminase activity to an extent similar to that with ATP, but without concomitanttransamidase activity and with a very low level of ATP-S hydrolysis. This uncoupling between ATP-Shydrolysis and glutaminase activities suggests that the activation of glutaminase activity by ATP or ATP-S, together with Glu-tRNAGln, results either from an allosteric effect due simply to binding of theseanalogues to the enzyme or from some structural changes that attend ATP or ATP-S hydrolysis.