Chock P B, Huang C Y, Timmons R B, Stadtman E R
Proc Natl Acad Sci U S A. 1973 Nov;70(11):3134-8. doi: 10.1073/pnas.70.11.3134.
A fluorescent derivative of ATP, epsilon-ATP, was used to adenylylate glutamine synthetase (EC 6.3.1.2) from Escherichia coli enzymatically. The epsilon-adenylylated enzyme exhibits similar catalytic properties and inhibitor susceptibility to those of the naturally adenylylated enzyme. The fluorescence properties of the epsilon-adenosine and of tryptophan residues of the enzyme were used to study ligand-induced conformational changes involving alterations of the tryptophan regions and the adenylylation site of the protein. Binding of Mn(2+) to the epsilon-adenylylated enzyme is accompanied by a decrease of epsilon-adenosine fluorescence as compared to the effect observed for the Mg(2+) binding. An ADP binding study shows that at low ADP concentration, ADP causes enhancement of the tryptophan fluorescence only, reflecting the binding to unadenylylated subunits; and at high ADP concentration, ADP causes not only enhancement of the fluorescence, but also a quenching of the fluorescence of enzyme-bound epsilon-AMP, reflecting binding to the adenylylated subunits. Dissociation constants calculated from these fluorescence changes agree well with those determined from binding studies of ADP to adenylylated and unadenylylated enzymes. Binding of the feedback inhibitor, alanine, to Mn(2+)-dependent glutamine synthetase causes enhancement of the epsilon-AMP fluorescence, from which a dissociation constant of 1.5 mM was calculated for the inhibitor. The fluorescence changes observed due to ligands binding suggest that Mg(2+) and Mn(2+) stabilize different conformational states of the enzyme.
ATP的一种荧光衍生物ε-ATP，被用于通过酶促反应将来自大肠杆菌的谷氨酰胺合成酶（EC 6.3.1.2）腺苷酰化。ε-腺苷酰化的酶表现出与天然腺苷酰化的酶相似的催化特性和对抑制剂的敏感性。利用该酶的ε-腺苷和色氨酸残基的荧光特性来研究配体诱导的构象变化，这些变化涉及蛋白质色氨酸区域和腺苷酰化位点的改变。与观察到的Mg(2+)结合的效果相比，Mn(2+)与ε-腺苷酰化的酶结合会伴随着ε-腺苷荧光的降低。一项ADP结合研究表明，在低ADP浓度下，ADP仅导致色氨酸荧光增强，这反映了其与未腺苷酰化亚基的结合；而在高ADP浓度下，ADP不仅导致荧光增强，还会淬灭酶结合的ε-AMP的荧光，这反映了其与腺苷酰化亚基的结合。根据这些荧光变化计算出的解离常数与通过ADP与腺苷酰化和未腺苷酰化酶的结合研究确定的解离常数非常吻合。反馈抑制剂丙氨酸与Mn(2+)依赖的谷氨酰胺合成酶结合会导致ε-AMP荧光增强，据此计算出该抑制剂的解离常数为1.5 mM。观察到的由于配体结合引起的荧光变化表明，Mg(2+)和Mn(2+)稳定了酶的不同构象状态。
