Abell L M, Villafranca J J
Department of Chemistry, Pennsylvania State University, University Park 16802.
Biochemistry. 1991 Jun 25;30(25):6135-41. doi: 10.1021/bi00239a008.
A number of slow tight-binding inhibitors are known for glutamine synthetase that resemble the geometry of the tetrahedral intermediate formed during the enzyme-catalyzed condensation of gamma-glutamyl phosphate and ammonia. One of these inhibitors, phosphinothricin [L-2-amino-4-(hydroxymethyl-phosphinyl)butanoic acid], has been investigated by rapid kinetic methods. Phosphinothricin not only exhibits the kinetic properties of a slow tight-binding inhibitor but also undergoes phosphorylation during the course of the ATP-dependent inactivation. The acid lability of phosphinothricin phosphate enabled investigation of the kinetics of glutamine synthetase inactivation using rapid quench kinetic techniques. The rate-limiting step in the inhibition reaction is the binding of inhibitor (0.004-0.014 microM-1 s-1) and/or a conformational change associated with binding, which is several orders of magnitude slower than the binding of ATP. The association rate of phosphinothricin depends on which metal ion is bound to the enzyme (Mn2+ or Mg2+). With Mn2+ bound to glutamine synthetase the rate of association and the phosphorylation rate are faster than when Mg2+ is bound. The data are interpreted with use of a model in which the binding of a substrate analogue with a tetrahedral moiety enhances the phosphorylation rate of the reaction intermediate; however, the initial binding interaction is retarded because the enzyme has to bind a molecule that has a "transition-state" geometry rather than a ground-state substrate structure. During the course of the inactivation, progressively slower rates for binding and phosphoryl transfer were observed, indicating communication between active sites.
已知有多种缓慢紧密结合型抑制剂作用于谷氨酰胺合成酶，它们类似于在酶催化的γ-谷氨酰磷酸与氨缩合过程中形成的四面体中间体的几何结构。其中一种抑制剂，草丁膦[L-2-氨基-4-(羟甲基膦酰基)丁酸]，已通过快速动力学方法进行了研究。草丁膦不仅表现出缓慢紧密结合型抑制剂的动力学特性，而且在ATP依赖性失活过程中会发生磷酸化。草丁膦磷酸盐的酸不稳定性使得能够使用快速淬灭动力学技术研究谷氨酰胺合成酶失活的动力学。抑制反应中的限速步骤是抑制剂的结合(0.004 - 0.014 μM-1 s-1)和/或与结合相关联的构象变化，这比ATP的结合慢几个数量级。草丁膦的缔合速率取决于与酶结合的是哪种金属离子(Mn2+ 或Mg2+)。当Mn(2+)与谷氨酰胺合成酶结合时，缔合速率和磷酸化速率比Mg(2+)结合时更快。数据是用一个模型来解释的，在该模型中，具有四面体部分的底物类似物的结合增强了反应中间体的磷酸化速率；然而，初始结合相互作用受到阻碍，因为酶必须结合一个具有“过渡态”几何结构而非基态底物结构的分子。在失活过程中，观察到结合和磷酰基转移的速率逐渐变慢，表明活性位点之间存在通讯。
