Identification by Mn2+ rescue of two residues essential for the proton transfer of tRNase Z catalysis.
Thermotoga maritima tRNase Z cleaves pre-tRNAs containing the 74CCA76 sequence precisely after the A76 residue to create the mature 3' termini. Its crystal structure has revealed a four-layer alphabeta/betaalpha sandwich fold that is typically found in the metallo-beta-lactamase superfamily. The well-conserved six histidine and two aspartate residues together with metal ions are assumed to form the tRNase Z catalytic center. Here, we examined tRNase Z variants containing single amino acid substitutions in the catalytic center for pre-tRNA cleavage. Cleavage by each variant in the presence of Mg2+ was hardly detected, although it is bound to pre-tRNA. Surprisingly, however, Mn2+ ions restored the lost Mg2+-dependent activity with two exceptions of the Asp52Ala and His222Ala substitutions, which abolished the activity almost completely. These results provide a piece of evidence that Asp-52 and His-222 directly contribute the proton transfer for the catalysis.
Minagawa
Asako
A
Department of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata 956-8603, Japan.
Takaku
Hiroaki
H
Ishii
Ryohei
R
Takagi
Masamichi
M
Yokoyama
Shigeyuki
S
Nashimoto
Masayuki
M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000737:chemistry; D001483:Base Sequence; D002384:Catalysis; D020134:Catalytic Domain; D002413:Cations, Divalent; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D008345:Manganese / Q000737:chemistry*; D008670:Metals / Q000737:chemistry; D008969:Molecular Sequence Data; D011522:Protons; D012322:RNA Precursors / Q000737:chemistry* / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry* / Q000378:metabolism; D020124:Thermotoga maritima / Q000201:enzymology
D002413:Cations, Divalent; D008670:Metals; D011522:Protons; D012322:RNA Precursors; D001224:Aspartic Acid; D008345:Manganese; D006639:Histidine; D012343:RNA, Transfer; D004722:Endoribonucleases