Trotta P P, Wellner V P, Pinkus L M, Meister A
Proc Natl Acad Sci U S A. 1973 Oct;70(10):2717-21. doi: 10.1073/pnas.70.10.2717.
Glutamine-dependent carbamylphosphate synthetase (Escherichia coli) is composed of a heavy subunit (molecular weight about 130,000) and a light subunit (molecular weight about 42,000), which can be separated with retention of catalytic activities. The separated heavy subunit can catalyze activation of CO(2) by ATP and synthesis of carbamylphosphate from ammonia (but not from glutamine). The only catalytic activity exhibited by the separated light subunit is the ability to hydrolyze glutamine; the separated heavy subunit does not exhibit glutaminase activity. The pH-activity curve of the glutaminase activity of native carbamylphosphate synthetase exhibits maxima at about pH 4.2 and 9.5, while the glutaminase activity of the separated light subunit exhibits only a single optimum at about pH 6.7. When the light and heavy subunits are recombined, the two pH optima characteristic of native enzyme are restored. Glutaminase activities of native enzyme at both pH optima are similarly inhibited by the glutamine analog, L-2-amino-4-oxo-5-chloropentanoic acid, and also by dithiothreitol. Storage of native enzyme at pH 9 abolishes the glutaminase optimum at acid pH, but greatly increases the activity at alkaline pH. Treatment of native enzyme with N-ethylmaleimide increases the glutaminase activity dramatically and converts the pH profile to one that closely resembles that of the isolated light subunit. The data indicate that the same active site is involved in hydrolysis of glutamine at both acid and alkaline values of pH, and that this property of the enzyme depends upon interactions between the heavy and light subunits of native enzyme. The double-optima behavior of native enzyme seems to be related to participation of different catalytic groups of the enzyme which affect the maximum velocity rather than the binding of substrate. The findings offer additional evidence for occurrence of significant interactions between the subunits of carbamylphosphate synthetase, and may have significance in relation to other glutamine amidotransferases, including glutaminases.
谷氨酰胺依赖性氨甲酰磷酸合成酶（大肠杆菌）由一个重亚基（分子量约130,000）和一个轻亚基（分子量约42,000）组成，二者可分离且保留催化活性。分离出的重亚基能催化ATP激活CO₂，并由氨（而非谷氨酰胺）合成氨甲酰磷酸。分离出的轻亚基唯一的催化活性是水解谷氨酰胺的能力；分离出的重亚基不表现出谷氨酰胺酶活性。天然氨甲酰磷酸合成酶的谷氨酰胺酶活性的pH-活性曲线在约pH 4.2和9.5处出现最大值，而分离出的轻亚基的谷氨酰胺酶活性仅在约pH 6.7处有一个最佳值。当轻亚基和重亚基重新组合时，天然酶特有的两个pH最佳值得以恢复。天然酶在两个pH最佳值处的谷氨酰胺酶活性同样受到谷氨酰胺类似物L-2-氨基-4-氧代-5-氯戊酸以及二硫苏糖醇的抑制。将天然酶保存在pH 9会消除酸性pH下的谷氨酰胺酶最佳值，但会大大增加碱性pH下的活性。用N-乙基马来酰亚胺处理天然酶会显著增加谷氨酰胺酶活性，并将pH谱转变为与分离出的轻亚基非常相似的谱。数据表明，在酸性和碱性pH值下，水解谷氨酰胺涉及相同的活性位点，并且该酶的这一特性取决于天然酶重亚基和轻亚基之间的相互作用。天然酶的双最佳行为似乎与影响最大速度而非底物结合的酶的不同催化基团的参与有关。这些发现为氨甲酰磷酸合成酶亚基之间存在显著相互作用提供了额外证据，并且可能与其他谷氨酰胺酰胺转移酶（包括谷氨酰胺酶）有关。
