title: Effects of neutral salts and pH on the activity and stability of human RNase H2.
abstract: 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 10-60 mM, 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 60-80 mM. The activity at 25-35 °C exhibited a narrow bell-shaped pH-dependence with the acidic and alkaline pKe (pKe1 and pKe2) values of 7.3 - 7.6 and 8.1 - 8.8, respectively. Enthalpy changes (ΔH°) of deprotonation were 5 ± 21 kJ mol-1 for pKe1 and 68 ± 25 kJ mol-1 for pKe2. These results suggest that the ionizable groups responsible for pKe1 may be two out of Asp34, Glu35 and Asp141 of DEDD motif, and that for pKe2 may be Lys69 of DSK motif.
authors.affiliation: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Oiwakecho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Misato
authors.initials: M
authors.lastname: Baba
authors.affiliation: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Oiwakecho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Kojima
authors.affiliation: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Oiwakecho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Rihoko
authors.initials: R
authors.lastname: Nakase
authors.affiliation: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Oiwakecho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Shota
authors.initials: S
authors.lastname: Imai
authors.affiliation: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Oiwakecho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Tomomi
authors.initials: T
authors.lastname: Yamasaki
authors.affiliation: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Oiwakecho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Teisuke
authors.initials: T
authors.lastname: Takita
authors.affiliation: Section on Formation of RNA, Division of Developmental Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Crouch
authors.affiliation: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Oiwakecho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Yasukawa
mesh_terms: D004305:Dose-Response Relationship, Drug; D004795:Enzyme Stability / Q000187:drug effects; D006801:Humans; D006863:Hydrogen-Ion Concentration; D016914:Ribonuclease H / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012492:Salts / Q000737:chemistry / Q000494:pharmacology*; D013329:Structure-Activity Relationship
chemical_list: D012492:Salts; C091139:ribonuclease HII; D016914:Ribonuclease H
keywords: activity; human RNase H2; neutral salt; pH dependence; stability