Trotta P P, Platzer K E, Haschemeyer R H, Meister A
Proc Natl Acad Sci U S A. 1974 Nov;71(11):4607-11. doi: 10.1073/pnas.71.11.4607.
In the course of studies on glutamine-dependent carbamyl phosphate synthetase from Aerobacter aerogenes, we purified another protein which was found to be glutamate synthase (EC 2.6.1.53). The enzyme, obtained in apparently homogeneous form (monomer molecular weight about 227,000; s(20,omega) = 17.6 S), was found to be a typical glutamine amidotransferase in that it exhibits glutaminase activity and can utilize ammonia in place of glutamine as a nitrogen donor. The enzyme also catalyzes at low rates the oxidative deamination of glutamate in the presence of TPN, and it exhibits TPNH oxidase activity. The enzyme is similar to the glutamate synthase found in Escherichia coli in that it is an iron-sulfide flavoprotein. Treatment of the enzyme with sodium dodecyl sulfate or potassium thiocyanate dissociates it into nonidentical subunits exhibiting molecular weights of about 175,000 and 51,500. The glutamine-dependent activity of the enzyme is inhibited by L-2-amino-4-oxo-5-chloropentanoic acid, but this chloroketone analog of glutamine does not affect the ammonia-dependent glutamate synthase activity. Studies with [(14)C]chloroketone show that the reagent binds to the heavy subunit only. Inhibition by the chloroketone and its binding to the heavy subunit are markedly reduced in the presence of L-glutamine. Sedimentation velocity studies carried out in potassium thiocyanate indicate that iron-sulfide and flavin sites are also located on the heavy subunit. While these studies show that glutamate synthase resembles other glutamine amidotransferases in certain of its catalytic properties, the findings indicate that the light subunit of this enzyme, in contrast to that of several other glutamine amidotransferases, does not function to bind glutamine. It is of interest that the enzyme exhibits an unusually high affinity for ammonia as compared to a number of other glutamine amidotransferases. Glutamate synthase is inhibited (competitively with respect to glutamine) by low concentrations of methionine sulfone, methionine sulfoximine, and methionine sulfoxide.
在对产气气杆菌中谷氨酰胺依赖性氨甲酰磷酸合成酶的研究过程中，我们纯化出了另一种蛋白质，结果发现它是谷氨酸合酶（EC 2.6.1.53）。该酶以明显均一的形式获得（单体分子量约为227,000；s(20,ω)=17.6 S），被发现是一种典型的谷氨酰胺氨基转移酶，因为它具有谷氨酰胺酶活性，并且可以利用氨替代谷氨酰胺作为氮供体。该酶在TPN存在的情况下也能以较低的速率催化谷氨酸的氧化脱氨基反应，并且具有TPNH氧化酶活性。该酶与在大肠杆菌中发现的谷氨酸合酶相似，因为它是一种铁硫黄素蛋白。用十二烷基硫酸钠或硫氰酸钾处理该酶会使其解离成分子量约为175,000和51,500的不同亚基。该酶的谷氨酰胺依赖性活性受到L-2-氨基-4-氧代-5-氯戊酸的抑制，但这种谷氨酰胺的氯酮类似物并不影响氨依赖性谷氨酸合酶的活性。用[(14)C]氯酮进行的研究表明，该试剂仅与重亚基结合。在L-谷氨酰胺存在的情况下，氯酮的抑制作用及其与重亚基的结合明显减弱。在硫氰酸钾中进行的沉降速度研究表明，铁硫和黄素位点也位于重亚基上。虽然这些研究表明谷氨酸合酶在某些催化特性上与其他谷氨酰胺氨基转移酶相似，但研究结果表明，与其他几种谷氨酰胺氨基转移酶相比，该酶的轻亚基不具有结合谷氨酰胺的功能。有趣的是，与许多其他谷氨酰胺氨基转移酶相比，该酶对氨表现出异常高的亲和力。低浓度的甲砜霉素、甲硫氨酸亚砜亚胺和甲硫氨酸亚砜会抑制谷氨酸合酶（相对于谷氨酰胺具有竞争性）。
