The interaction of the trp repressor from Escherichia coli with L-tryptophan and indole propanoic acid.
The interaction of the trp repressor from Escherichia coli with L-tryptophan and indole propanoic acid.
复制标题
大肠杆菌色氨酸阻遏物与 L-色氨酸和吲哚丙酸的相互作用。
DOI：
10.1111/j.1432-1033.1986.tb09682.x
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发表时间：
1986
期刊：
影响因子：
--
通讯作者：
Lane,AN
中科院分区：
文献类型：
--
作者：
Lane,AN
利用吸光度、荧光、圆二色性和质子核磁共振光谱研究了大肠杆菌中辅酶抑制物l -色氨酸和诱导剂吲哚丙酸与辅酶抑制物的结合。这两种配体以相同的方向结合到阻遏物上的相同位点；它们是分子上的竞争者。结合位点极性相对较低，包含至少一个位于结合配体C5质子附近的吲哚部分上方0.3 nm处的甲基，以及一个芳香残基，可能是酪氨酸。解离常数随温度和pH的变化而变化。在25°C、0.1 M、pH 7.6的磷酸盐缓冲液中，两种配体的解离常数均为18±2 μM。在同一缓冲体系中，色氨酸的范霍夫解离焓为35.5±1 kJ/mol，吲哚丙酸的范霍夫解离焓为30.5±2 kJ/mol。抑制因子对吲哚丙酸的亲和力在7 < pH < 10范围内与pH无关，但对色氨酸的亲和力在相同范围内降低了4倍。色氨酸的氨基对其结合亲和力有重要贡献。差异核磁共振谱显示结合配体上的蛋白质共振变化不大。用差谱法和核奥氏效应谱法对结合共振的核磁共振信号进行了赋值。结合共振的性质与配体在结合位点内大部分固定一致。两种配体的光谱差异和已知的功能差异表明，色氨酸在抑制物中诱导的构象状态与吲哚丙酸诱导的构象状态略有不同。没有证据表明会发生全球性的转变。配体的解离速率相对较大，在400-600 s−1范围内。
The binding of the corepressor,l‐tryptophan, and an inducer, indole propanoic acid, to the trp repressor fromEscherichia coliwas studied by absorbance, fluorescence, circular dichroic and proton NMR spectroscopy. The two ligands bind to the same site on the repressor in the same orientation; they are molecular competitors. The binding site is of relatively low polarity and contains at least one methyl group that lies 0.3 nm over the indole moiety near the C5 proton of the bound ligand, and an aromatic residue, probably tyrosine. The dissociation constant was determined as a function of temperature and pH. At 25°C in 0.1 M phosphate buffer, pH 7.6, the dissociation constant is 18 ± 2 μM for both ligands. In the same buffer system, the van't Hoff enthalpy for dissociation is 35.5 ± 1 kJ/mol for tryptophan, and 30.5 ± 2 kJ/mol for indole propanoic acid. The affinity of the repressor for indole propanoic acid is independent of pH in the range 7 < pH < 10, but decreases four fold for tryptophan in the same range. The amino group of tryptophan makes a significant contribution to its binding affinity. Difference NMR spectra showed that there are few changes of protein resonances on binding ligands. The NMR signals of the bound resonances were assigned by difference and nuclear Overhauser effect spectroscopy. The properties of the bound resonances are consistent with the ligands being largely immobilised within the binding site. The difference spectra, and the known functional differences of the two ligands, suggest that tryptophan induces a slightly different conformational state in the repressor from that induced by indole propanoic acid. There is no evidence for a global transition. The rate of dissociation of ligands is relatively large, being in the range 400–600 s−1.