Effects of neutral salts and pH on the activity and stability of human RNase H2

Ribonuclease H (RNase H) specifically degrades the RNA of RNA/DNA hybrid. Recent study has shown that a single ribonucleotide is embedded in DNA double strand at every few thousand base pairs in human genome, and human RNase H2 is involved in its removal. Here, we examined the effects of neutral salts and pH on the activity and stability of human RNase H2. NaCl, KCl, RbCl and NaBr increased the activity to 170-390% at 10-60mM, while LiCl, LiBr and CsCl inhibited it, suggesting that species of cation, but not anion, is responsible for the effect on activity. NaCl and KCl increased the stability by decreasing the first-order rate constant of the inactivation to 50-60% at 60-80mM. The activity at 25-35 degrees C exhibited a narrow bell-shaped pH-dependence with the acidic and alkaline pKe (pK(e1) and pK(e2)) values of 7.3-7.6 and 8.1-8.8, respectively. Enthalpy changes (Delta H degrees) of deprotonation were 5 +/- 21 kJ mol(-1) for pK(e1) and 68 +/- 25kJ mol(-1) for pK(e2). These results suggest that the ionizable groups responsible for pK(e1) may be two out of Asp34, Glu35 and Asp141 of DEDD motif, and that for pK(e2) may be Lys69 of DSK motif.