Casey C A, Anderson P M
J Biol Chem. 1983 Jul 25;258(14):8723-32.
The glutamine- and N-acetyl-L-glutamate-dependent carbamoyl phosphate synthetase III present in liver of largemouth bass (Micropterus salmoides) has been highly purified. The properties of the enzyme are generally similar to the properties of the carbamoyl phosphate synthetase III from spiny dogfish (Squalus acanthias) previously described (Anderson, P. M. (1981) J. Biol. Chem. 256, 12228-12238). However, the bass enzyme is not subject to self-association, and the effects of urea and, particularly, trimethylamine-N-oxide, on catalytic activity are considerably reduced. Ammonia can substitute for glutamine as the nitrogen-donating substrate, but the maximum rate is lower. Carbamoyl phosphate synthetase III, like other carbamoyl phosphate synthetases, catalyzes two partial reactions, ATP synthesis from carbamoyl phosphate and ADP, and bicarbonate-dependent hydrolysis of ATP; both reactions are greatly stimulated by the presence of N-acetyl-L-glutamate. Carbamoyl phosphate synthetase III gave no detectable immunological cross-reaction with antibody to the ammonia- and N-acetyl-L-glutamate-dependent carbamoyl phosphate synthetase from rat liver mitochondria. The apparent Km value for N-acetyl-L-glutamate decreases significantly as the concentration of L-glutamine increases in the glutamine-dependent reaction, and vice versa. This effect is glutamine-specific. The apparent Km for N-acetyl-L-glutamate in the ammonia-dependent reaction is not affected by changes in ammonia concentration and the apparent Km for ammonia (8 mM) is also not affected by changes in N-acetyl-L-glutamate concentration. Studies involving inhibition of carbamoyl phosphate synthetase III by the glutamine analogs acivicin (L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid), DON (6-diazo-5-oxo-L-norleucine), and chloroketone (L-2-amino-4-oxo-5-chloropentanoic acid), provided additional evidence for significant interaction between the L-glutamine- and N-acetyl-L-glutamate-binding sites. Glutamine-dependent but not ammonia-dependent activity is inhibited by preincubating the enzyme with these analogs. This inhibition requires the presence of both MgATP and N-acetyl-L-glutamate, and is prevented by the additional presence of L-glutamine. Inhibition of the glutamine-dependent reaction by DON or chloroketone is accompanied by a decrease in the apparent Km for N-acetyl-L-glutamate in the ammonia-dependent reaction from 0.3 mM to a value which is nearly the same as that observed in the glutamine-dependent reaction when glutamine is saturating (0.015 mM).
大口黑鲈（Micropterus salmoides）肝脏中存在的谷氨酰胺和N - 乙酰 - L - 谷氨酸依赖性氨甲酰磷酸合成酶III已被高度纯化。该酶的性质与先前描述的棘鲨（Squalus acanthias）的氨甲酰磷酸合成酶III的性质总体相似（Anderson，P.M.（1981）J. Biol. Chem. 256，12228 - 12238）。然而，鲈鱼的这种酶不会发生自我缔合，并且尿素，尤其是三甲胺 - N - 氧化物对催化活性的影响显著降低。氨可以替代谷氨酰胺作为供氮底物，但最大反应速率较低。氨甲酰磷酸合成酶III与其他氨甲酰磷酸合成酶一样，催化两个部分反应，即由氨甲酰磷酸和ADP合成ATP，以及ATP的碳酸氢盐依赖性水解；这两个反应在N - 乙酰 - L - 谷氨酸存在时均受到极大刺激。氨甲酰磷酸合成酶III与大鼠肝线粒体中氨和N - 乙酰 - L - 谷氨酸依赖性氨甲酰磷酸合成酶的抗体没有可检测到的免疫交叉反应。在谷氨酰胺依赖性反应中，随着L - 谷氨酰胺浓度的增加，N - 乙酰 - L - 谷氨酸的表观Km值显著降低，反之亦然。这种效应是谷氨酰胺特异性的。在氨依赖性反应中，N - 乙酰 - L - 谷氨酸的表观Km不受氨浓度变化的影响，氨的表观Km（8 mM）也不受N - 乙酰 - L - 谷氨酸浓度变化的影响。涉及谷氨酰胺类似物阿西维辛（L -（αS，5S） - α - 氨基 - 3 - 氯 - 4,5 - 二氢 - 5 - 异恶唑乙酸）、DON（6 - 重氮 - 5 - 氧代 - L - 正亮氨酸）和氯酮（L - 2 - 氨基 - 4 - 氧代 - 5 - 氯戊酸）对氨甲酰磷酸合成酶III抑制作用的研究，为L - 谷氨酰胺和N - 乙酰 - L - 谷氨酸结合位点之间的显著相互作用提供了额外证据。用这些类似物预孵育该酶会抑制谷氨酰胺依赖性而非氨依赖性活性。这种抑制需要MgATP和N - 乙酰 - L - 谷氨酸同时存在，并且L - 谷氨酰胺的额外存在可阻止这种抑制。DON或氯酮对谷氨酰胺依赖性反应的抑制伴随着氨依赖性反应中N - 乙酰 - L - 谷氨酸的表观Km从0.3 mM降至一个与谷氨酰胺饱和时谷氨酰胺依赖性反应中观察到的值（0.015 mM）几乎相同的值。
