Lysyl residue 47 is near the subunit ATP-binding site of glutamine synthetase from Escherichia coli.
Lysyl residue 47 is near the subunit ATP-binding site of glutamine synthetase from Escherichia coli.
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赖氨酰残基 47 靠近大肠杆菌谷氨酰胺合成酶的亚基 ATP 结合位点。
DOI：
10.1016/s0021-9258(17)42745-1
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发表时间：
1984
期刊：
影响因子：
--
通讯作者：
R. Heinrikson
中科院分区：
文献类型：
--
作者：
H. Pinkofsky;A. Ginsburg;I. Reardon;R. Heinrikson
ATP类似物5'-对氟磺酰基苯甲酰腺苷（FSBA）灭活大肠杆菌中的十二聚体谷氨酰胺合成酶，同时标记一个位点/亚基(Foster， W.B, Griffith, m.j., and Kingdon， H.S.（1981）。化学，246,882-886)。溴化氰在70%的HCOOH中裂解fsba -灭活酶，产生一个大肽（Mr等于4600），产率约为75%，含有N ε -4-羧基苯磺酰（CBS）赖氨酸。cbs肽在246 nm处具有较高的紫外吸光度（δ ε等于19,000 M-1 cm-1），采用高效液相色谱法对其进行纯化。标记肽的出现与从未修饰的活性酶中丢失的肽（缺乏酪氨酸和色氨酸）相一致；cbs肽也可以与含有半胱氨酸的巯基硝基苯甲酸标记肽和腺苷化肽区分开。当未腺苷化酶在没有或存在Mn2+或Mg2+ （+/- l -谷氨酸）的情况下用FSBA灭活，或酶被腺苷化时，标记相同的肽。因此，这些酶之间的构象差异不能通过ATP位点的亲和标记来检测。fsba标记的肽从亚基多肽链的n端跨越9-48个残基，通过自动Edman降解的序列分析显示，cbs -赖氨酸位于47号位置。因此，赖氨酸残基47似乎靠近天然谷氨酰胺合成酶结构中ATP的亚基γ -磷酸结合位点。给出了cbs标记肽的一级结构。
The ATP analog 5'-p-fluorosulfonylbenzoyladenosine (FSBA) inactivates dodecameric glutamine synthetase from Escherichia coli with concomitant labeling of one site/subunit (Foster, W.B., Griffith, M.J., and Kingdon, H.S. (1981) J. Biol. Chem. 246, 882-886). Cyanogen bromide cleavage of the FSBA-inactivated enzyme in 70% HCOOH produced a large peptide (Mr congruent to 4600) in approximately 75% yield containing N epsilon-4-carboxybenzenesulfonyl (CBS) lysine. The CBS-peptide was purified by high performance liquid chromatography on the basis of its relatively high UV absorbance at 246 nm (delta epsilon congruent to 19,000 M-1 cm-1). The appearance of the labeled peptide coincided with the loss of a peptide (lacking tyrosine and tryptophan) from the unmodified, active enzyme; the CBS-peptide also could be distinguished from cysteine-containing peptides labeled with thionitrobenzoate and from the adenylylated peptide. The identical peptide was labeled when the unadenylylated enzyme was inactivated with FSBA in the absence or presence of Mn2+ or Mg2+ (+/- L-glutamate) or when the enzyme was adenylylated. Conformational differences among these enzyme forms therefore are not detected by affinity labeling of ATP sites. The FSBA-labeled peptide spans residues 9-48 from the N-terminal end of the subunit polypeptide chain, and sequence analysis by automated Edman degradation revealed that CBS-lysine was at position 47. Thus, lysyl residue 47 appears to be near the subunit gamma-phosphate-binding site for ATP in the native glutamine synthetase structure. The primary structure of the CBS-labeled peptide is presented.