EPSILON-ADENYLATED GLUTAMINE SYNTHETASE - INTERNAL FLUORESCENCE PROBE FOR ENZYME CONFORMATION
EPSILON-ADENYLATED GLUTAMINE SYNTHETASE - INTERNAL FLUORESCENCE PROBE FOR ENZYME CONFORMATION
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DOI：
10.1073/pnas.70.11.3134
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发表时间：
1973-01-01
影响因子：
11.1
通讯作者：
STADTMAN, ER
中科院分区：
文献类型：
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作者：
CHOCK, PB;HUANG, CY;STADTMAN, ER
利用ATP的荧光衍生物ε-ATP对大肠杆菌的谷氨酰胺合成酶（EC 6.3.1.2）进行酶水解。ε-腺苷化酶表现出与天然腺苷化酶相似的催化性能和抑制剂敏感性。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，包括色氨酸区域和蛋白质腺苷化位点的改变。与Mg2+结合观察到的效果相比，Mn2+与ε-腺苷化酶的结合伴随着ε-腺苷荧光的减少。一项ADP结合研究表明，在低ADP浓度下，ADP仅引起色氨酸荧光增强，反映了与未腺苷化亚基的结合；在高ADP浓度下，ADP不仅使荧光增强，而且使酶结合的ε-AMP荧光猝灭，反映了与腺苷化亚基的结合。从这些荧光变化计算的解离常数与从ADP与腺苷化酶和非腺苷化酶的结合研究中确定的解离常数一致。反馈抑制剂丙氨酸与依赖于Mn2+的谷氨酰胺合成酶结合可增强ε-AMP荧光，由此计算出该抑制剂的解离常数为1.5 mM。由于配体结合而观察到的荧光变化表明Mg2+和Mn2+稳定了酶的不同构象状态。
A fluorescent derivative of ATP, ε-ATP, was used to adenylylate glutamine synthetase (EC 6.3.1.2) fromEscherichia colienzymatically. The ε-adenylylated enzyme exhibits similar catalytic properties and inhibitor susceptibility to those of the naturally adenylylated enzyme. The fluorescence properties of the ε-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 Mn2+to the ε-adenylylated enzyme is accompanied by a decrease of ε-adenosine fluorescence as compared to the effect observed for the Mg2+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 ε-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 Mn2+-dependent glutamine synthetase causes enhancement of the ε-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 Mg2+and Mn2+stabilize different conformational states of the enzyme.