An engineered blockage within the ammonia tunnel of carbamoyl phosphate synthetase prevents the use of glutamine as a substrate but not ammonia
An engineered blockage within the ammonia tunnel of carbamoyl phosphate synthetase prevents the use of glutamine as a substrate but not ammonia
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DOI：
10.1021/bi9926173
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发表时间：
2000-03-28
期刊：
影响因子：
2.9
通讯作者：
Raushel, FM
中科院分区：
文献类型：
--
作者：
Huang, XY;Raushel, FM
大肠杆菌的异二聚氨甲酰磷酸合成酶（CPS）催化碳酸氢盐、谷氨酰胺和两个ATP分子形成氨甲酰磷酸。该酶催化小酰胺转移酶亚基内谷氨酰胺的水解，然后将氨转移到大亚基内的两个活性位点。这三个活性位点通过分子间隧道连接，该隧道位于来自E.杆菌已经提出氨中间体通过该分子通道从小亚基内的谷氨酰胺结合位点扩散到大亚基内的碳酸氢盐磷酸化位点。为了提供这种分子隧道的功能意义的实验支持，限定小亚基内的"氨隧道"的内壁的残基被靶向定点诱变。这些结构修饰旨在阻止或阻碍氨向大亚基的传递。两个突变蛋白（G359 Y和G359 F）显示动力学特性与氨通道的收缩或阻塞一致。对于这两种突变体，转氨酶和碳酸氢盐依赖性ATP酶反应已经彼此解偶联。然而，这些突变体酶是完全功能时，外部氨被用作氮源，但不能使用谷氨酰胺合成氨甲酰-P。这些结果表明存在一个替代途径的碳酸氢盐磷酸化位点时，氨作为外部氮源提供。
The heterodimeric carbamoyl phosphate synthetase (CPS) from Escherichia coli catalyzes the formation of carbamoyl phosphate from bicarbonate, glutamine, and two molecules of ATP. The enzyme catalyzes the hydrolysis of glutamine within the small amidotransferase subunit and then transfers ammonia to the two active sites within the large subunit. These three active sites are connected via an intermolecular tunnel, which has been located within the X-ray crystal structure of CPS from E. coli. It has been proposed that the ammonia intermediate diffuses through this molecular tunnel from the binding site for glutamine within the small subunit to the phosphorylation site for bicarbonate within the large subunit. To provide experimental support for the functional significance of this molecular tunnel, residues that define the interior walls of the "ammonia tunnel" within the small subunit were targeted for site-directed mutagenesis. These structural modifications were intended to either block or impede the passage of ammonia toward the large subunit. Two mutant proteins (G359Y and G359F) display kinetic properties consistent with a constriction or blockage of the ammonia tunnel. With both mutants, the glutaminase and bicarbonate-dependent ATPase reactions have become uncoupled from one another. However, these mutant enzymes are fully functional when external ammonia is utilized as the nitrogen source but are unable to use glutamine for the synthesis of carbamoyl-P. These results suggest the existence of an alternate route to the bicarbonate phosphorylation site when ammonia is provided as an external nitrogen source.