Affinity labeling of hepatitis C virus replicase with a nucleotide analogue: identification of binding site.
Affinity labeling of hepatitis C virus replicase with a nucleotide analogue: identification of binding site.
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DOI：
10.1021/bi301098g
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发表时间：
2013-01-15
期刊：
影响因子：
2.9
通讯作者：
Pandey VN
中科院分区：
文献类型：
--
作者：
Manvar D;Singh K;Pandey VN
我们使用了一个ATP类似物，5‘-[p-(氟磺酰)苯甲酰基]腺苷(FSBA)来修饰丙型肝炎病毒复制酶，以确定酶中的ATP结合部位。FSBA以浓度依赖的方式灭活丙型肝炎病毒复制酶的活性，结合化学计量比为每摩尔酶2摩尔FSBA。在存在rNTP底物或双链RNA模板引物的情况下，酶活性不受FSBA的影响，这些底物或双链RNA模板引物不支持ATP作为传入核苷酸，但在PolyrU.rA26存在的情况下不受FSBA的影响。对FSBA修饰酶的胰蛋白酶多肽进行了高效液相色谱分析，发现在23min和36min有两个不同的多肽被洗脱，而对照组没有。此外，我们还注意到这两种多肽都不受Mg.ATP存在的FSBA修饰的影响。对从高效液相色谱法中纯化的亲和标记的胰多肽进行LC/MS/MS分析，发现在382位(Tyr)和491位(Lys)有两个主要的修饰位点，在38位(Tyr)有一个次要的修饰位点。为了验证Tyr38、Tyr382和Lys491在催化中的功能意义，我们分别用丙氨酸取代了这些残基，并检测了它们催化RdRp活性的能力。我们发现Y382A型和K491A型突变体对RdRp的催化活性都有明显的影响，而Y38A型没有受到影响。我们进一步观察到，Y382a和K491a突变体与模板引物的结合能力没有受到影响，但在没有或存在模板引物的情况下，它们对ATP的光交联能力受到了显著影响。
We have used an ATP analog, 5′-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA), to modify HCV replicase in order to identify ATP binding site in the enzyme. FSBA inactivates HCV replicase activity in a concentration dependent manner with a binding stoichiometry of 2 moles of FSBA per mole of enzyme. The enzyme activity is protected from FSBA in the presence of rNTP substrates or double stranded RNA template primers that do not support ATP as the incoming nucleotide but not in the presence of polyrU.rA26. The HPLC analysis of tryptic peptides of FSBA-modified enzyme revealed the presence of two distinct peptides eluted at 23 min and 36 min; these were absent in the control. Further we noted that both the peptides were protected from FSBA modification in the presence of Mg.ATP. The LC/MS/MS analysis of the affinity-labeled tryptic peptides purified from HPLC, identified two major modification sites at positions 382 (Tyr), and 491 (Lys) and a minor site at position 38 (Tyr). To validate the functional significance of Tyr38, Tyr382 and Lys491 in catalysis, we individually substituted these residues by alanine and examined their ability to catalyze RdRp activity. We found that both Y382A and K491A mutants were significantly affected in their ability to catalyze RdRp activity while Y38A remained unaffected. We further observed that both Y382A and K491A mutants were not affected in their ability to bind template primer but significantly affected in their ability to photo-crosslink ATP in the absence or presence of template primer.
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影响因子：
5.4
作者：
Bressanelli, S;Tomei, L;De Francesco, R
通讯作者：
De Francesco, R
影响因子：
3.7
作者：
Hong, Z;Cameron, CE;Zhong, WD
通讯作者：
Zhong, WD
影响因子：
4.8
作者：
Bougie, I;Bisaillon, M
通讯作者：
Bisaillon, M
DOI：
10.1073/pnas.89.21.10011
发表时间：
1992-11-01
影响因子：
11.1
作者：
CHIEN, DY;CHOO, QL;KUO, G
通讯作者：
KUO, G
影响因子：
5.4
作者：
Chinnaswamy, S.;Murali, A.;Kao, C. C.
通讯作者：
Kao, C. C.