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 Mn(2+)-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 Mg(2+) or Mn(2+) ions. We observed that the Mn(2+) ions cannot rescue the activities of Asp(I)Ala and His(V)Ala variants from each species, which are lost in the presence of Mg(2+). This observation may support the proposed catalytic mechanism.
Elbarbary
Reyad A
RA
Department of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata, Niigata 956-8603, Japan.
Takaku
Hiroaki
H
Nashimoto
Masayuki
M
D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000737:chemistry* / Q000235:genetics; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain / Q000502:physiology; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006639:Histidine / Q000737:chemistry* / Q000235:genetics; D008345:Manganese / Q000737:chemistry*; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D001224:Aspartic Acid; D008345:Manganese; D006639:Histidine; D004722:Endoribonucleases