Anderson P M, Carlson J D
Biochemistry. 1975 Aug 12;14(16):3688-94. doi: 10.1021/bi00687a027.
Carbamyl phosphate synthetase from Escherichia coli reacts stoichiometrically (one to one) with [14C]cyanate to give a 14C-labeled complex which can be isolated by gel filtration. The formation of the complex is prevented if L-glutamine is present or if the enzyme is first reacted with 2-amino-4-oxo-5-chloropentanoic acid, a chloro ketone analog of glutamine which has been shown to react with a specific SH group in the glutamine binding site. The rate of complex formation is increased by ADP and decreased by ATP and HCO3-. The isolated complex is inactive with respect to glutamine-dependent synthetase activity. However, the reaction of cyanate with the enzyme is reversible. The rate of dissociation of the isolated complex is not affected by pH (over the pH range 6-10), is greatly increased by ATP and HCO3-, and is decreased by ADP. The allosteric effectors ornithine and UMP have no effect on either the rate of formation or the rate of dissociation of the complex; however, the apparent affinity of the enzyme for ATP is decreased by UMP and increased by ornithine. The site of reaction of cyanate with carbamyl phosphate synthetase, which is composed of a light and a heavy subunit, is with an SH group in the light subunit to give an S-carbamylcysteine residue. The binding of L-[14C]glutamine to the enzyme and the inhibition of glutamine-dependent synthetase activity by the chloroketone analog are both prevented by the presence of cyanate. The reaction with cyanate is considered to be with the same essential SH group which is located in the glutamine binding site and is alkylated by 2-amino-4-oxo-5-chloropentanoic acid. The bicarbonate-dependent effects of ATP suggest that formation of the activated carbon dioxide intermediate is accompanied by changes in the heavy subunit which functionally alter the properties of the glutamine binding site on the light subunit. The allosteric effects of ornithine and UMP are probably not related to this intersubunit interaction.
来自大肠杆菌的氨甲酰磷酸合成酶与[14C]氰酸盐按化学计量比（一对一）反应，生成一种可通过凝胶过滤分离的14C标记复合物。如果存在L-谷氨酰胺，或者该酶先与2-氨基-4-氧代-5-氯戊酸（谷氨酰胺的氯酮类似物，已证明其可与谷氨酰胺结合位点中的特定SH基团反应）反应，则复合物的形成会受到抑制。ADP可提高复合物的形成速率，而ATP和HCO3-则会降低该速率。分离得到的复合物对谷氨酰胺依赖性合成酶活性无活性。然而，氰酸盐与该酶的反应是可逆的。分离得到的复合物的解离速率不受pH（6-10的pH范围内）影响，ATP和HCO3-可使其显著增加，而ADP则使其降低。变构效应物鸟氨酸和UMP对复合物的形成速率或解离速率均无影响；然而，UMP会降低该酶对ATP的表观亲和力，而鸟氨酸则会增加该亲和力。氰酸盐与由轻链和重链亚基组成的氨甲酰磷酸合成酶的反应位点是在轻链亚基中的一个SH基团上，生成一个S-氨甲酰半胱氨酸残基。氰酸盐的存在可阻止L-[14C]谷氨酰胺与该酶的结合以及氯酮类似物对谷氨酰胺依赖性合成酶活性的抑制。与氰酸盐的反应被认为是与位于谷氨酰胺结合位点且被2-氨基-4-氧代-5-氯戊酸烷基化的相同必需SH基团发生反应。ATP对碳酸氢盐的依赖性效应表明，活性二氧化碳中间体的形成伴随着重链亚基的变化，这些变化在功能上改变了轻链亚基上谷氨酰胺结合位点的性质。鸟氨酸和UMP的变构效应可能与这种亚基间相互作用无关。
