Binding of nucleotides by T4 DNA ligase and T4 RNA ligase: Optical absorbance and fluorescence studies
Binding of nucleotides by T4 DNA ligase and T4 RNA ligase: Optical absorbance and fluorescence studies
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DOI：
10.1016/s0006-3495(01)75985-1
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发表时间：
2001-12-01
影响因子：
3.4
通讯作者：
de Vries, S
中科院分区：
文献类型：
--
作者：
Cherepanov, AV;de Vries, S
用紫外-可见吸收光谱和荧光光谱表征了核苷酸与T4DNA和RNA连接酶的相互作用。这两种酶都将核苷酸与Kd在0.1到20微米之间结合。核苷酸结合导致260 nm处的吸光度下降，这是由于与芳香族残基，可能是苯丙氨酸的圆周率堆积，并且由于与外环氨基的氢键，导致最大吸光度红移。T4DNA连接酶除了具有催化的ATP结合部位外，还有另一个非共价核苷酸结合部位。结合在那里的ATP改变了核苷酸在催化位置的pi堆积，增加了它的光学消光。非共价位的K类似于催化位的1000倍。核苷酸猝灭蛋白质的荧光，表明色氨酸残基位于连接酶的活性部位。在298 nm附近的吸光度的下降表明该色氨酸残基在连接酶-核苷酸复合体中的氢键作用被削弱。T4RNA连接酶的激发/发射特性表明，它的ATP结合口袋与溶剂接触，而溶剂在与核苷酸结合时被排除。总体而言，光谱分析揭示了T4连接酶和相关核苷酸转移酶之间的重要相似之处，尽管序列相似性较低。
The interaction of nucleotides with T4 DNA and RNA ligases has been characterized using ultraviolet visible (UV-VIS) absorbance and fluorescence spectroscopy. Both enzymes bind nucleotides with the Kd between 0.1 and 20 muM. Nucleotide binding results in a decrease of absorbance at 260 nm due to pi -stacking with an aromatic residue, possibly phenylalanine, and causes red-shifting of the absorbance maximum due to hydrogen bonding with the exocyclic amino group. T4 DNA ligase is shown to have, besides the catalytic ATP binding site, another noncovalent nucleotide binding site. ATP bound there alters the pi -stacking of the nucleotide in the catalytic site, increasing its optical extinction. The K. for the noncovalent site is similar to 1000-fold higher than for the catalytic site. Nucleotides quench the protein fluorescence showing that a tryptophan residue is located in the active site of the ligase. The decrease of absorbance around 298 nm suggests that the hydrogen bonding interactions of this tryptophan residue are weakened in the ligase-nucleotide complex. The excitation/emission properties of T4 RNA ligase indicate that its ATP binding pocket is in contact with solvent, which is excluded upon binding of the nucleotide. Overall, the spectroscopic analysis reveals important similarities between T4 ligases and related nucleotidyltransferases, despite the low sequence similarity.