Beschreibung:
<jats:p>Ribonuclease <jats:styled-content style="fixed-case">H</jats:styled-content>1 is a conserved enzyme that cleaves the RNA strand of <jats:styled-content style="fixed-case">RNA</jats:styled-content>·<jats:styled-content style="fixed-case">DNA</jats:styled-content> heteroduplexes and has important functions in the nuclear and mitochondrial compartments. The therapeutic action of antisense oligodeoxynucleotides involves the recruitment of <jats:styled-content style="fixed-case">RN</jats:styled-content>ase <jats:styled-content style="fixed-case">H</jats:styled-content>1 to cleave disease‐relevant <jats:styled-content style="fixed-case">RNA</jats:styled-content> targets. Recombinant human (<jats:styled-content style="fixed-case">H</jats:styled-content>s) <jats:styled-content style="fixed-case">RN</jats:styled-content>ase <jats:styled-content style="fixed-case">H</jats:styled-content>1 was purified from a bacterial expression host, and conditions were identified that provided optimal oligonucleotide‐directed <jats:styled-content style="fixed-case">RNA</jats:styled-content> cleavage <jats:italic>in vitro</jats:italic>. <jats:styled-content style="fixed-case">H</jats:styled-content>s‐<jats:styled-content style="fixed-case">RN</jats:styled-content>ase <jats:styled-content style="fixed-case">H</jats:styled-content>1 exhibits optimal catalytic activity in <jats:styled-content style="fixed-case">pH</jats:styled-content> 7.5 <jats:styled-content style="fixed-case">HEPES</jats:styled-content> buffer and a salt (<jats:styled-content style="fixed-case">KC</jats:styled-content>l) concentration of ~ 100–150 m<jats:sc>m</jats:sc>. <jats:styled-content style="fixed-case">M</jats:styled-content>g<jats:sup>2+</jats:sup> best supports <jats:styled-content style="fixed-case">H</jats:styled-content>s‐<jats:styled-content style="fixed-case">RN</jats:styled-content>ase <jats:styled-content style="fixed-case">H</jats:styled-content>1 with an optimal concentration of 10 m<jats:sc>m</jats:sc>, but at higher concentrations inhibits enzyme activity. <jats:styled-content style="fixed-case">M</jats:styled-content>n<jats:sup>2+</jats:sup> and <jats:styled-content style="fixed-case">C</jats:styled-content>o<jats:sup>2+</jats:sup> also support catalytic activity, while <jats:styled-content style="fixed-case">N</jats:styled-content>i<jats:sup>2+</jats:sup> and <jats:styled-content style="fixed-case">Z</jats:styled-content>n<jats:sup>2+</jats:sup> exhibit only modest activities as cofactors. The optimized assay was used to show that an antisense oligonucleotide, added in substoichiometric amounts to initiate <jats:styled-content style="fixed-case">RNA</jats:styled-content> cleavage, supports up to 30 rounds of reaction in 30 min. Mutation to alanine of the conserved histidine at position 264 causes an ~ 100‐fold decrease in <jats:italic>k</jats:italic><jats:sub>cat</jats:sub> under multiple‐turnover conditions, but does not alter <jats:italic>K</jats:italic><jats:sub>m</jats:sub>. Under single‐turnover conditions, the <jats:styled-content style="fixed-case">H</jats:styled-content>264<jats:styled-content style="fixed-case">A</jats:styled-content> mutant exhibits a 12‐fold higher exponential time constant for substrate cleavage. The defective activity of the <jats:styled-content style="fixed-case">H</jats:styled-content>264<jats:styled-content style="fixed-case">A</jats:styled-content> mutant is not rescued in either assay condition by higher <jats:styled-content style="fixed-case">M</jats:styled-content>g<jats:sup>2+</jats:sup> concentrations. These data implicate the <jats:styled-content style="fixed-case">H</jats:styled-content>264 side chain in phosphodiester hydrolysis as well as in product release, and are consistent with a proposed model in which the <jats:styled-content style="fixed-case">H</jats:styled-content>264 side chain interacts with a divalent metal ion to support catalysis.</jats:p>