Abstract
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.