Rubino S D, Nyunoya H, Lusty C J
J Biol Chem. 1987 Mar 25;262(9):4382-6.
The cloned carAB operon of Escherichia coli coding for the small and large subunits of carbamyl phosphate synthetase has been used to construct a recombinant plasmid with a 4.16 kilobase ClaI fragment of the car operon that lacks the major promoters, P1 and P2. The plasmid, pHN12, carries a functional carB gene. A mutant E. coli strain lacking both subunits of carbamyl phosphate synthetase when transformed with pHN12 overproduces the large subunit by 200-fold (8-10% of the cellular protein). The elevated levels of the large subunit enable the transformed cells to utilize NH3 but not glutamine as nitrogen donor for carbamyl phosphate synthesis. The large subunit has been purified from the overexpressing strain. The purified native large subunit is capable of synthesizing carbamyl phosphate from ammonia, HCO-3, and ATP. The kinetic properties of the large subunit compared with the holoenzyme indicate that the Michaelis constants of the large subunit for HCO-3 and ATP are modulated by its association with the small glutamine binding subunit.
编码氨甲酰磷酸合成酶小亚基和大亚基的大肠杆菌克隆carAB操纵子已被用于构建一个重组质粒，该质粒含有car操纵子的一个4.16千碱基的ClaI片段，该片段缺乏主要启动子P1和P2。质粒pHN12携带一个功能性的carB基因。当用pHN12转化时，缺乏氨甲酰磷酸合成酶两个亚基的大肠杆菌突变株会使大亚基过量产生200倍（占细胞蛋白质的8 - 10%）。大亚基水平的升高使转化细胞能够利用NH3而不是谷氨酰胺作为氨甲酰磷酸合成的氮供体。大亚基已从过表达菌株中纯化出来。纯化的天然大亚基能够从氨、HCO-3和ATP合成氨甲酰磷酸。与全酶相比，大亚基的动力学特性表明，大亚基对HCO-3和ATP的米氏常数受其与小的谷氨酰胺结合亚基的结合调节。
