Val143 of human ribonuclease H2 is not critical for, but plays a role in determining catalytic activity and substrate specificity
Val143 of human ribonuclease H2 is not critical for, but plays a role in determining catalytic activity and substrate specificity
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DOI：
10.1371/journal.pone.0228774
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发表时间：
2020-02
期刊：
影响因子：
3.7
通讯作者：
Misato Baba;K. Kojima;Takuto Nishimura;Takuya Sugiura;T. Takita;R. Uehara;R. Crouch;K. Yasukawa
中科院分区：
文献类型：
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作者：
Misato Baba;K. Kojima;Takuto Nishimura;Takuya Sugiura;T. Takita;R. Uehara;R. Crouch;K. Yasukawa
核糖核酸酶H2 （RNase H2）具有单核糖核苷酸切除活性（活性A）和RNA链降解活性（活性B）。人RNase H2的Val143位于活性位点，在真核RNase H2中保守。在这项研究中，我们探讨了Val143在催化活性和底物特异性中的作用。在大肠杆菌中表达了19个氨基酸位置143的单变异体，除V143C和V143M外，其余变异体均从细胞中纯化。当野生型人RNase H2 （WT）的活性设置为100%时，17个变异的相对活性A和B分别在0.05 ~ 130和0.02 ~ 42%之间。当WT的相对活性A与相对活性B之比设为1时，17个变异体的比值在0.2 ~ 5.7之间。这表明缬氨酸是平衡这两种活性的最佳选择。V143Y和V143W的比值相对较高（分别为5.6和5.5），表明143位的酪氨酸和色氨酸等大块残基对可剪切磷酸二酯键5′侧的核糖核苷酸糖段的2′-OH造成了位阻。V143Q的比值相对较低（0.2）。这些结果表明，Val143在决定催化活性和底物特异性方面起作用，而不是至关重要的。
Ribonuclease H2 (RNase H2) exhibits both single ribonucleotide excision activity (activity A) and RNA strand degrading activity (activity B). Val143 of human RNase H2 is located at the active site and is conserved in eukaryotic RNase H2. In this study, we explored the role of Val143 in catalytic activity and substrate specificity. Nineteen single variants at amino acid position 143 were expressed in E. coli, and all variants except for V143C and V143M were purified from the cells. When the activity of the wild-type human RNase H2 (WT) was set as 100%, the relative activities A and B of the 17 variants were in the range of 0.05–130 and 0.02–42%, respectively. When the ratio of the relative activity A to the relative activity B of WT was set as 1, the ratios of the 17 variants were in the range of 0.2–5.7. This indicates that valine is optimal for balancing the two activities. The ratios for V143Y and V143W were relatively high (5.6 and 5.5, respectively), suggesting that the bulky residues like tyrosine and tryptophan at position 143 caused steric hindrance with the 2’-OH of the sugar moiety of the ribonucleotide at the 5’ side of the scissile phosphodiester bond. The ratio for V143Q was relatively low (0.2). These results suggested that Val143 is not critical for, but plays a role in determining catalytic activity and substrate specificity.