Functional analyses for tRNase Z variants: An aspartate and a histidine in the active site are essential for the catalytic activity

We performed functional analyses for various single amino-acid substitution variants of Escherichia coli, Bacillus subtilis, and human tRNase Zs. The well-conserved six histidine, His(I)-His(VI), and two aspartate, Asp(I) and Asp(II), residues together with metal ions are thought to form the active site of tRNase Z. The Mn²⁺-rescue analysis for Thermotoga maritima tRNase Z^S has suggested that Asp(I) and His(V) directly contribute the proton transfer for the catalysis, and a catalytic mechanism has been proposed. However, experimental evidence supporting the proposed mechanism was limited. Here we intensively examined E. coli and B. subtilis tRNase Z^S variants and human tRNase Z^L variants for cleavage activities on pre-tRNAs in the presence of Mg²⁺ or Mn²⁺ ions. We observed that the Mn²⁺ ions cannot rescue the activities of Asp(I)Ala and His(V)Ala variants from each species, which are lost in the presence of Mg²⁺. This observation may support the proposed catalytic mechanism.