Späth Bettina, Settele Florian, Schilling Oliver, D'Angelo Igor, Vogel Andreas, Feldmann Ingo, Meyer-Klaucke Wolfram, Marchfelder Anita
Molekulare Botanik, Universität Ulm, Albert-Einstein-Allee 11, 89069 Ulm, Germany.
Biochemistry. 2007 Dec 25;46(51):14742-50. doi: 10.1021/bi7010459. Epub 2007 Dec 4.
The endonuclease tRNase Z from A. thaliana (AthTRZ1) was originally isolated for its tRNA 3' processing activity. Here we show that AthTRZ1 also hydrolyzes the phosphodiester bond in bis(p-nitrophenyl) phosphate (bpNPP) with a kcat of 7.4 s-1 and a KM of 8.5 mM. We analyzed 22 variants of AthTRZ1 with respect to their ability to hydrolyze bpNPP. This mutational mapping identified fourteen variants that lost the ability to hydrolyze bpNPP and seven variants with reduced activity. Surprisingly, a single amino acid change (R252G) resulted in a ten times higher activity compared to the wild type enzyme. tRNase Z enzymes exist in long and short forms. We show here that in contrast to the short tRNase Z enzyme AthTRZ1, the long tRNase Z enzymes do not have bpNPP hydrolysis activity pointing to fundamental differences in substrate cleavage between the two enzyme forms. Furthermore, we determined the metal content of AthTRZ1 and analyzed the metal requirement for bpNPP hydrolysis. AthTRZ1 shows a high affinity for Zn2+ ions; even upon incubation with metal chelators, 0.76 Zn2+ ions are retained per dimer. In contrast to bpNPP hydrolysis, pre-tRNA processing requires additional metal ions, Mn2+ or Mg2+, as Zn2+ ions alone are insufficient.
来自拟南芥的核酸内切酶tRNase Z（AthTRZ1）最初因其tRNA 3'加工活性而被分离出来。在此我们表明，AthTRZ1还能水解双（对硝基苯基）磷酸酯（bpNPP）中的磷酸二酯键，其催化常数kcat为7.4 s-1，米氏常数KM为8.5 mM。我们分析了AthTRZ1的22个变体水解bpNPP的能力。这种突变图谱鉴定出14个丧失水解bpNPP能力的变体和7个活性降低的变体。令人惊讶的是，单个氨基酸变化（R252G）导致活性比野生型酶高10倍。tRNase Z酶以长形式和短形式存在。我们在此表明，与短tRNase Z酶AthTRZ1不同，长tRNase Z酶不具有bpNPP水解活性，这表明两种酶形式在底物切割方面存在根本差异。此外，我们测定了AthTRZ1的金属含量，并分析了bpNPP水解对金属的需求。AthTRZ1对Zn2+离子具有高亲和力；即使与金属螯合剂孵育，每个二聚体仍保留0.76个Zn2+离子。与bpNPP水解不同，前体tRNA加工需要额外的金属离子，Mn2+或Mg2+，因为仅Zn2+离子是不够的。
