Abdelal A T, Ingraham J L
J Biol Chem. 1975 Jun 25;250(12):4410-7.
Carbamylphosphate synthetase was purified to homogeneity from a derepressed strain of Salmonella typhimurium by a procedure based on affinity chromatography employing immobilized glutamine. The enzyme catalyzes the synthesis of carbamylphosphate from either ammonia or glutamine together with ATP and bicarbonate. The ATP saturation curve of either nitrogen donor is sigmoidal (n equals 1.5) but the affinity for ATP is higher with ammonia. In addition to the feedback inhibition by UMP and activation by ornithine which we previously reported (1), the activity was found to be stimulated by IMP and phosphoribosyl-1-pyrophosphate. Evidence from pool measurements in enteric bacteria by others suggests that of the latter two compounds only phosphoribosyl-1-pyrophosphate is physiologically significant. All effectors regulate enzyme activity by altering its affinity for ATP. Glutamine also modulates the affinity for ATP; it is increased as glutamine concentratiions decrease, an effect that could serve to insulate the cell against major changes in carbamylphosphate synthesis in response to fluctuations in concentration of glutamine. The molecular weight of the holoenzyme was estimated to be 150,000 by sucrose density gradient centrifugation in triethanolamine and Tris-acetate buffers in which the enzyme is a monomer. In the presence of ornithine in potassium phosphate buffer, the enzyme is an oligomer with a molecular weight of 580,000. This transition has been exploited as an alternate route of purifying the enzyme to homogeneity using successive sucrose density centrifugation. Polyacrylamide gel electrophoresis of the enzyme in the presence of sodium dodecyl sulfate shows that the enzyme consists of two unequal subunits with molecular weights of 110,000 and 45,000. The two subunits were separated by gel filtration in the presence of 1 M potassium thiocyanate, ATP, MgCl2, glutamine, NH4Cl, ornithine, and UMP. The heavy subunit catalyzes the synthesis of carbamylphosphate from ammonia but not glutamine. The ATP saturation curve for the separated heavy subunit is still sigmoidal (n equals 1.4 and So.5 equals 0.3 mM). The ammonia dependent activity of the heavy subunit is stimulated by the activators ornithine, IMP, and phosphoribosyl-1-pyrophosphate but is only marginally inhibited by high concentrations of UMP. The addition of the light subunit restored full ability to utilize glutamine as well as normal sensitivity to UMP. Purified subunits were used for in vitro complementation studies with strains carrying mutations in pyrA, the structural gene encoding carbamylphosphate synthetase. The results indicate that the pyrA region encodes both subunits and that the structural genes for the two polypeptides are linked. A deletion mutant lacking both subunits of carbamylphosphate synthetase also lacked any ability to synthetize carbamylphosphate from ammonia. Hence, unlike certain other bacteria, S. typhimurium does not possess a carbamate kinase.
通过基于使用固定化谷氨酰胺的亲和层析方法，从鼠伤寒沙门氏菌的去阻遏菌株中纯化出了均一的氨甲酰磷酸合成酶。该酶催化由氨或谷氨酰胺与ATP和碳酸氢盐合成氨甲酰磷酸。两种氮供体的ATP饱和曲线均为S形（n等于1.5），但氨对ATP的亲和力更高。除了我们之前报道的UMP反馈抑制和鸟氨酸激活（1）外，还发现IMP和磷酸核糖-1-焦磷酸可刺激该酶的活性。其他人对肠道细菌中代谢物池的测量证据表明，后两种化合物中只有磷酸核糖-1-焦磷酸具有生理意义。所有效应物都通过改变其对ATP的亲和力来调节酶活性。谷氨酰胺也调节对ATP的亲和力；随着谷氨酰胺浓度降低，其亲和力增加，这种效应可使细胞免受因谷氨酰胺浓度波动而导致的氨甲酰磷酸合成的重大变化影响。在三乙醇胺和Tris-乙酸盐缓冲液中，通过蔗糖密度梯度离心法估计全酶的分子量为150,000，在该缓冲液中酶为单体。在磷酸钾缓冲液中存在鸟氨酸时，该酶为分子量为580,000的寡聚体。利用这种转变，通过连续蔗糖密度离心法作为将酶纯化至均一的另一种途径。在十二烷基硫酸钠存在下对该酶进行聚丙烯酰胺凝胶电泳表明，该酶由分子量分别为110,000和45,000的两个不等亚基组成。在1 M硫氰酸钾、ATP、MgCl2、谷氨酰胺、NH4Cl、鸟氨酸和UMP存在下，通过凝胶过滤将两个亚基分离。重亚基催化由氨而非谷氨酰胺合成氨甲酰磷酸。分离出的重亚基的ATP饱和曲线仍然是S形（n等于1.4，So.5等于0.3 mM）。重亚基的氨依赖性活性受到激活剂鸟氨酸、IMP和磷酸核糖-1-焦磷酸的刺激，但仅受到高浓度UMP的轻微抑制。添加轻亚基恢复了利用谷氨酰胺的全部能力以及对UMP的正常敏感性。使用纯化的亚基对携带氨甲酰磷酸合成酶结构基因pyrA突变的菌株进行体外互补研究。结果表明，pyrA区域编码两个亚基，并且这两个多肽的结构基因是连锁的。缺乏氨甲酰磷酸合成酶两个亚基的缺失突变体也完全丧失了由氨合成氨甲酰磷酸的能力。因此，与某些其他细菌不同，鼠伤寒沙门氏菌不具有氨基甲酸激酶。
