Maurizi M R, Pinkofsky H B, Ginsburg A
Section on Protein Chemistry, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892.
Biochemistry. 1987 Aug 11;26(16):5023-31. doi: 10.1021/bi00390a021.
The binding of divalent cations and nucleotide to bovine brain glutamine synthetase and their effects on the activity of the enzyme were investigated. In ADP-supported gamma-glutamyl transfer at pH 7.2, kinetic analyses of saturation functions gave [S]0.5 values of approximately 1 microM for Mn2+, approximately 2 mM for Mg2+, 19 nM for ADP.Mn, and 7.2 microM for ADP.Mg. The method of continuous variation applied to the Mn2+-supported reaction indicated that all subunits of the purified enzyme express activity when 1.0 equiv of ADP is bound per subunit. Measurements of equilibrium binding of Mn2+ to the enzyme in the absence and presence of ADP were consistent with each subunit binding free Mn2+ (KA approximately equal to 1.5 X 10(5) M-1) before binding the Mn.ADP complex (KA' approximately equal to 1.1 X 10(6) M-1). The binding of the first Mn2+ or Mg2+ to each subunit produces structural perturbations in the octameric enzyme, as evidenced by UV spectral and tryptophanyl residue fluorescence changes. The enzyme, therefore, has one structural site per subunit for Mn2+ or Mg2+ and a second site per subunit for the metal ion-nucleotide complex, both of which must be filled for activity expression. Chloride binding (KA' approximately equal to 10(4) M-1) to the enzyme was found to have a specific effect on the protein conformation, producing a substantial (30%) quench of tryptophanyl fluorescence and increasing the affinity of the enzyme 2-4-fold for Mg2+ or Mn2+. Arsenate, which activates the gamma-glutamyl transfer activity by binding to an allosteric site, and L-glutamate also cause conformational changes similar to those produced by Cl- binding. Anion binding to allosteric sites and divalent metal ion binding at active sites both produce tryptophanyl residue exposure and tyrosyl residue burial without changing the quaternary enzyme structure.
研究了二价阳离子和核苷酸与牛脑谷氨酰胺合成酶的结合及其对该酶活性的影响。在pH 7.2的ADP支持的γ-谷氨酰转移反应中，对饱和函数进行动力学分析得出，对于Mn2+，[S]0.5值约为1 μM；对于Mg2+，约为2 mM；对于ADP·Mn，为19 nM；对于ADP·Mg，为7.2 μM。应用连续变化法对Mn2+支持的反应进行分析表明，当每个亚基结合1.0当量的ADP时，纯化酶的所有亚基均表现出活性。在不存在和存在ADP的情况下，对酶与Mn2+的平衡结合进行测量，结果表明每个亚基在结合Mn·ADP复合物（KA'约等于1.1×10(6) M-1）之前先结合游离Mn2+（KA约等于1.5×10(5) M-1）。每个亚基结合第一个Mn2+或Mg2+会在八聚体酶中产生结构扰动，紫外光谱和色氨酸残基荧光变化证明了这一点。因此，该酶每个亚基有一个用于Mn2+或Mg2+的结构位点，以及每个亚基有第二个用于金属离子-核苷酸复合物的位点，两者都必须被占据才能表达活性。发现氯离子与该酶的结合（KA'约等于10(4) M-1）对蛋白质构象有特定影响，使色氨酸荧光大幅淬灭（30%），并使酶对Mg2+或Mn2+的亲和力增加2至4倍。砷酸盐通过结合变构位点激活γ-谷氨酰转移活性，L-谷氨酸也会引起与氯离子结合产生的构象变化类似的变化。阴离子与变构位点的结合以及二价金属离子在活性位点的结合都会导致色氨酸残基暴露和酪氨酸残基埋藏，而不会改变酶的四级结构。
