Liaw S H, Kuo I, Eisenberg D
Institute of Molecular Medicine, School of Medicine, National Taiwan University, Taipei.
Protein Sci. 1995 Nov;4(11):2358-65. doi: 10.1002/pro.5560041114.
Glutamine synthetase (GS) catalyzes the ATP-dependent condensation of ammonia and glutamate to yield glutamine, ADP, and inorganic phosphate in the presence of divalent cations. Bacterial GS is an enzyme of 12 identical subunits, arranged in two rings of 6, with the active site between each pair of subunits in a ring. In earlier work, we have reported the locations within the funnel-shaped active site of the substrates glutamate and ATP and of the two divalent cations, but the site for ammonia (or ammonium) has remained elusive. Here we report the discovery by X-ray crystallography of a binding site on GS for monovalent cations, Tl+ and Cs+, which is probably the binding site for the substrate ammonium ion. Fourier difference maps show the following. (1) Tl+ and Cs+ bind at essentially the same site, with ligands being Glu 212, Tyr 179, Asp 50', Ser 53' of the adjacent subunit, and the substrate glutamate. From its position adjacent to the substrate glutamate and the cofactor ADP, we propose that this monovalent cation site is the substrate ammonium ion binding site. This proposal is supported by enzyme kinetics. Our kinetic measurements show that Tl+, Cs+, and NH4+ are competitive inhibitors to NH2OH in the gamma-glutamyl transfer reaction. (2) GS is a trimetallic enzyme containing two divalent cation sites (n1, n2) and one monovalent cation site per subunit. These three closely spaced ions are all at the active site: the distance between n1 and n2 is 6 A, between n1 and Tl+ is 4 A, and between n2 and Tl+ is 7 A. Glu 212 and the substrate glutamate are bridging ligands for the n1 ion and Tl+. (3) The presence of a monovalent cation in this site may enhance the structural stability of GS, because of its effect of balancing the negative charges of the substrate glutamate and its ligands and because of strengthening the "side-to-side" intersubunit interaction through the cation-protein bonding. (4) The presence of the cofactor ADP increases the Tl+ binding to GS because ADP binding induces movement of Asp 50' toward this monovalent cation site, essentially forming the site. This observation supports a two-step mechanism with ordered substrate binding: ATP first binds to GS, then Glu binds and attacks ATP to form gamma-glutamyl phosphate and ADP, which complete the ammonium binding site. The third substrate, an ammonium ion, then binds to GS, and then loses a proton to form the more active species ammonia, which attacks the gamma-glutamyl phosphate to yield Gln. (5) Because the products (Glu or Gln) of the reactions catalyzed by GS are determined by the molecule (water or ammonium) attacking the intermediate gamma-glutamyl phosphate, this negatively charged ammonium binding pocket has been designed naturally for high affinity of ammonium to GS, permitting glutamine synthesis to proceed in aqueous solution.
谷氨酰胺合成酶（GS）在二价阳离子存在的情况下，催化氨和谷氨酸的ATP依赖性缩合反应，生成谷氨酰胺、ADP和无机磷酸。细菌GS是一种由12个相同亚基组成的酶，这些亚基排列成两个由6个亚基组成的环，活性位点位于环中每对亚基之间。在早期的工作中，我们已经报道了底物谷氨酸和ATP以及两个二价阳离子在漏斗形活性位点内的位置，但氨（或铵离子）的结合位点仍然难以确定。在此，我们报告通过X射线晶体学发现GS上的一价阳离子（Tl⁺和Cs⁺）结合位点，该位点可能是底物铵离子的结合位点。傅里叶差值图显示如下：（1）Tl⁺和Cs⁺基本上结合在同一位置，配体为相邻亚基的Glu 212、Tyr 179、Asp 50'、Ser 53'以及底物谷氨酸。从其与底物谷氨酸和辅因子ADP相邻位置来看，我们认为这个一价阳离子位点是底物铵离子结合位点。这一推测得到酶动力学的支持。我们的动力学测量表明，在γ-谷氨酰转移反应中，Tl⁺、Cs⁺和NH₄⁺是NH₂OH的竞争性抑制剂。（2）GS是一种三金属酶，每个亚基含有两个二价阳离子位点（n1、n2）和一个一价阳离子位点。这三个紧密相邻的离子都位于活性位点：n1和n2之间的距离为6 Å，n1和Tl⁺之间的距离为4 Å，n2和Tl⁺之间的距离为7 Å。Glu 212和底物谷氨酸是n1离子和Tl⁺的桥连配体。（3）该位点存在一价阳离子可能会增强GS的结构稳定性，这是因为它平衡了底物谷氨酸及其配体的负电荷，并且通过阳离子 - 蛋白质键加强了“侧向”亚基间相互作用。（4）辅因子ADP的存在增加了Tl⁺与GS的结合，因为ADP的结合诱导Asp 50'向这个一价阳离子位点移动，基本上形成了该位点。这一观察结果支持了有序底物结合的两步机制：ATP首先与GS结合，然后Glu结合并攻击ATP形成γ-谷氨酰磷酸和ADP，从而完成铵结合位点的形成。第三个底物铵离子随后与GS结合，然后失去一个质子形成活性更高的氨，氨攻击γ-谷氨酰磷酸生成谷氨酰胺。（5）由于GS催化反应的产物（谷氨酸或谷氨酰胺）取决于攻击中间产物γ-谷氨酰磷酸的分子（水或铵），这个带负电荷的铵结合口袋自然设计为对铵具有高亲和力，从而使谷氨酰胺合成能够在水溶液中进行。
