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Literature summary for 2.7.7.72 extracted from
- Pyrophosphorolysis of CCA addition: implication for fidelity (2011), J. Mol. Biol., 414, 28-43.
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| overexpression of C-terminally His-tagged enzyme in Escherichia coli | Escherichia coli |
| overexpression of C-terminally His-tagged enzyme in Escherichia coli | Homo sapiens |
| overexpression of C-terminally His-tagged enzyme in Escherichia coli | Archaeoglobus fulgidus |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | kinetic parameters of diphosphorolysis from A76, C75, and C74, overview | Escherichia coli | |
| 0.5 | - |
diphosphate | pH not specified in the publication, 37°C, diphosphorolysis reaction with substrate tRNA-C75 | Escherichia coli |  |
| 0.6 | - |
diphosphate | pH not specified in the publication, 37°C, diphosphorolysis reaction with substrate tRNA-C74 | Escherichia coli | |
| 1 | - |
diphosphate | pH not specified in the publication, 37°C, diphosphorolysis reaction with substrate tRNA-A76 | Escherichia coli | |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Mg2+ | essentially required, two metal ions are coordinated by highly conserved carboxylates and fulfill specific roles in catalyzing the reaction. Metal ion A activates the 3'-hydroxyl group of the primer for a nucleophilic in-line attack on the alpha-phosphate of the incoming NTP, while metal ion B promotes the leaving of the diphosphate group that is released during this reaction | Archaeoglobus fulgidus | |
| Mg2+ | essentially required, two metal ions are coordinated by highly conserved carboxylates and fulfill specific roles in catalyzing the reaction. Metal ion A activates the 3'-hydroxyl group of the primer for a nucleophilic in-line attack on the alpha-phosphate of the incoming NTP, while metal ion B promotes the leaving of the diphosphate group that is released during this reaction. The Mg2+ ions are also required for the diposphorolysis reaction, overview | Homo sapiens | |
| Mg2+ | essentially required, two metal ions are coordinated by highly conserved carboxylates and fulfill specific roles in catalyzing the reaction. Metal ion A activates the 3'-hydroxyl group of the primer for a nucleophilic in-line attack on the alpha-phosphate of the incoming NTP, while metal ion B promotes the leaving of the diphosphate group that is released during this reaction. The Mg2+ ions are also required for the diposphorolysis reaction, overview. With tRNA-A76 as the substrate in the reverse reacction, only Mg2+ is catalytically competent | Escherichia coli | |
| Mn2+ | while Mn2+ is incompetent for diphosphorolysis of tRNA-A76, it promotes a more rapid forward synthesis of tRNA-A76 than even the Mg2+ ion | Escherichia coli | |
| additional information | the nature of the divalent metal ions can influence the positioning of diphosphate in the active site | Homo sapiens | |
| additional information | the nature of the divalent metal ions can influence the positioning of diphosphate in the active site | Archaeoglobus fulgidus | |
| additional information | the nature of the divalent metal ions can influence the positioning of diphosphate in the active site. tRNAs ending in C75 and C74, in contrast to tRNA A76, are also active with other divalent metal ions tan Mg2+, albeit less efficient due to accumulation of unreacted tRNA substrate. Diphosphorolysis of tRNA-C75 proceeds the farthest with Mg2+, followed by Co2+, and by Mn2+ and Ni2+. Pyrophosphorolysis of tRNA-C74 also proceeds the farthest with Mg2+, but it is followed by Mn2+ and Co2+ and followed by Ni2+. While the preference of divalent metal ions varies among the three reactions, it also differs from that required for the forward A76 addition. While Ca2+ and Pb2+ fail to promote diphosphorolysis of all three tRNA substrates, they also fail to promote forward synthesis of tRNA-A76 for EcCCA | Escherichia coli | |
| Zn2+ | it is inert for diphosphorolysis of tRNAA76, but it is active in the forward synthesis of this tRNA | Escherichia coli | |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| a tRNA precursor + 2 CTP + ATP | Escherichia coli | - |
a tRNA with a 3' CCA end + 3 diphosphate | - |
r | |
| a tRNA precursor + 2 CTP + ATP | Homo sapiens | - |
a tRNA with a 3' CCA end + 3 diphosphate | - |
r | |
| a tRNA with a 3' CCA end + 3 diphosphate | Escherichia coli | - |
a tRNA precursor + 2 CTP + ATP | - |
r | |
| a tRNA with a 3' CCA end + 3 diphosphate | Homo sapiens | - |
a tRNA precursor + 2 CTP + ATP | - |
r | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Archaeoglobus fulgidus | - |
- |
- |
| Escherichia coli | - |
- |
- |
| Homo sapiens | - |
- |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant C-terminally His-tagged enzyme from Escherichia coli | Escherichia coli |
| recombinant C-terminally His-tagged enzyme from Escherichia coli | Homo sapiens |
| recombinant C-terminally His-tagged enzyme from Escherichia coli | Archaeoglobus fulgidus |
Source Tissue
| Source Tissue | Comment | Organism | Textmining |
|---|---|---|---|
| cell culture | optimal growth condition of the organismat 83°C | Archaeoglobus fulgidus | - |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| a tRNA precursor + 2 CTP + ATP | - |
Escherichia coli | a tRNA with a 3' CCA end + 3 diphosphate | - |
r | |
| a tRNA precursor + 2 CTP + ATP | - |
Homo sapiens | a tRNA with a 3' CCA end + 3 diphosphate | - |
r | |
| a tRNA precursor + 2 CTP + ATP | substrate is synthetic DNA templates based on the sequence of Escherichia coli tRNAVal. Overall reaction | Archaeoglobus fulgidus | a tRNA with a 3' CCA end + 3 diphosphate | - |
r | |
| a tRNA precursor + 2 CTP + ATP | substrate is synthetic DNA templates based on the sequence of Escherichia coli tRNAVal. Overall reaction, class II CCA-adding enzymes also perform the reverse reaction, mechanism, overview. The enzyme catalyzes diphosphorolysis slowly relative to the forward nucleotide addition and that it exhibits weak binding affinity to diphosphate relative to NTP | Escherichia coli | a tRNA with a 3' CCA end + 3 diphosphate | - |
r | |
| a tRNA precursor + 2 CTP + ATP | substrate is synthetic DNA templates based on the sequence of Escherichia coli tRNAVal. Overall reaction, class II CCA-adding enzymes also perform the reverse reaction, mechanism, overview. The enzyme catalyzes diphosphorolysis slowly relative to the forward nucleotide addition and that it exhibits weak binding affinity to diphosphate relative to NTP | Homo sapiens | a tRNA with a 3' CCA end + 3 diphosphate | - |
r | |
| a tRNA with a 3' CCA end + 3 diphosphate | - |
Escherichia coli | a tRNA precursor + 2 CTP + ATP | - |
r | |
| a tRNA with a 3' CCA end + 3 diphosphate | - |
Homo sapiens | a tRNA precursor + 2 CTP + ATP | - |
r | |
| a tRNA with a 3' CCA end + 3 diphosphate | substrate is synthetic DNA templates based on the sequence of Escherichia coli tRNAVal. Overall reaction, class II CCA-adding enzymes also perform the reverse reaction, mechanism, overview. The enzyme catalyzes diphosphorolysis slowly relative to the forward nucleotide addition and that it exhibits weak binding affinity to diphosphate relative to NTP | Escherichia coli | a tRNA precursor + 2 CTP + ATP | - |
r | |
| a tRNA with a 3' CCA end + 3 diphosphate | substrate is synthetic DNA templates based on the sequence of Escherichia coli tRNAVal. Overall reaction, class II CCA-adding enzymes also perform the reverse reaction, mechanism, overview. The enzyme catalyzes diphosphorolysis slowly relative to the forward nucleotide addition and that it exhibits weak binding affinity to diphosphate relative to NTP | Homo sapiens | a tRNA precursor + 2 CTP + ATP | - |
r | |
| additional information | class I CCA enzymes do not catalyze diphosphorolysis in contrast to class II CCA enzymes, overview | Archaeoglobus fulgidus | ? | - |
? | |
| additional information | only class II CCA enzymes catalyze diphosphorolysis, the reaction can initiate from all three CCA positions and proceed processively until the removal of nucleotide C74. Diphosphorolysis enables class II enzymes to efficiently remove an incorrect A75 nucleotide from the 3' end, at a rate much faster than the rate of A75 incorporation, suggesting the ability to perform a previously unexpected quality control mechanism for CCA synthesis. No activity with non-tRNA substrate BMVTLSTyr or U2 snRNA. The enzyme shows a robust activity with tRNA-A75, degrading it down to tRNA-A73 (by 50%) while showing a minor activity with tRNA-C76 (less than 5% substrate conversion) and no activity with tRNA-A74. The incorrect A75 is more readily removed than it is synthesized, suggesting a quality control mechanism that can improve the overall accuracy of CCA synthesis | Homo sapiens | ? | - |
? | |
| additional information | only class II CCA enzymes catalyze diphosphorolysis, the reaction can initiate from all three CCA positions and proceed processively until the removal of nucleotide C74. Diphosphorolysis enables class II enzymes to efficiently remove an incorrect A75 nucleotide from the 3' end, at a rate much faster than the rate of A75 incorporation, suggesting the ability to perform a previously unexpected quality control mechanism for CCA synthesis. No activity with non-tRNA substrate U2 snRNA, but EcCCA is active with non-tRNA substrate BMV TLSTyr and removes the terminalA nucleotide without proceeding further. The enzyme shows a robust activity with tRNA-A75, degrading it down to tRNA-A73 (by 50%) while showing a minor activity with tRNA-C76 (less than 5% substrate conversion) and no activity with tRNA-A74. The incorrect A75 is more readily removed than it is synthesized, suggesting a quality control mechanism that can improve the overall accuracy of CCA synthesis | Escherichia coli | ? | - |
? | |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| CCA enzyme | - |
Escherichia coli |
| CCA enzyme | - |
Homo sapiens |
| CCA enzyme | - |
Archaeoglobus fulgidus |
| class I CCA | - |
Archaeoglobus fulgidus |
| class I CCA enzyme | - |
Archaeoglobus fulgidus |
| class II CCA | - |
Escherichia coli |
| class II CCA | - |
Homo sapiens |
| class II CCA enzyme | - |
Escherichia coli |
| class II CCA enzyme | - |
Homo sapiens |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 37 | - |
assay at | Escherichia coli |
| 37 | - |
assay at | Homo sapiens |
| 55 | - |
assay at | Archaeoglobus fulgidus |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 1.4 | - |
diphosphate | pH not specified in the publication, 37°C, diphosphorolysis reaction with substrate tRNA-A76 | Escherichia coli | |
| 1.9 | - |
diphosphate | pH not specified in the publication, 37°C, diphosphorolysis reaction with substrate tRNA-C75 | Escherichia coli | |
| 3.1 | - |
diphosphate | pH not specified in the publication, 37°C, diphosphorolysis reaction with substrate tRNA-C74 | Escherichia coli | |
General Information
| General Information | Comment | Organism |
|---|---|---|
| evolution | diphosphorolysis of class II enzymes establishes a fundamental difference from class I enzymes, and it is achieved only with the tRNA structure and with specific divalent metal ions | Escherichia coli |
| evolution | diphosphorolysis of class II enzymes establishes a fundamental difference from class I enzymes, and it is achieved only with the tRNA structure and with specific divalent metal ions | Homo sapiens |
| evolution | diphosphorolysis of class II enzymes establishes a fundamental difference from class I enzymes, and it is achieved only with the tRNA structure and with specific divalent metal ions | Archaeoglobus fulgidus |
| additional information | the CCA enzymes are unusual RNA polymerases, which catalyze CCA addition to positions 74-76 at the tRNA 3' end without using a nucleic acid template, reaction mechanism of CCA addition and reverse phosphorolysis reaction, overview | Escherichia coli |
| additional information | the CCA enzymes are unusual RNA polymerases, which catalyze CCA addition to positions 74-76 at the tRNA 3' end without using a nucleic acid template, reaction mechanism of CCA addition and reverse phosphorolysis reaction, overview | Homo sapiens |
| additional information | the CCA enzymes are unusual RNA polymerases, which catalyze CCA addition to positions 74-76 at the tRNA 3' end without using a nucleic acid template, reaction mechanism of CCA addition, overview | Archaeoglobus fulgidus |
| physiological function | CCA enzymes catalyze stepwise CCA addition to the tRNA 3' end at positions 74--76 as an obligatory sequence for tRNA activity in the cell | Archaeoglobus fulgidus |
| physiological function | CCA enzymes catalyze stepwise CCA addition to the tRNA 3' end at positions 74-76 as an obligatory sequence for tRNA activity in the cell. Only class II CCA enzymes catalyze pyrophosphorolysis, the reaction can initiate from all three CCA positions and proceed processively until the removal of nucleotide C74. Diphosphorolysis enables class II enzymes to efficiently remove an incorrect A75 nucleotide from the 3' end, at a rate much faster than the rate of A75 incorporation, suggesting the ability to perform a previously unexpected quality control mechanism for CCA synthesis | Homo sapiens |
| physiological function | CCA enzymes catalyze stepwise CCA addition to the tRNA 3' end at positions 7476 as an obligatory sequence for tRNA activity in the cell. Only class II CCA enzymes catalyze pyrophosphorolysis, the reaction can initiate from all three CCA positions and proceed processively until the removal of nucleotide C74. Diphosphorolysis enables class II enzymes to efficiently remove an incorrect A75 nucleotide from the 3' end, at a rate much faster than the rate of A75 incorporation, suggesting the ability to perform a previously unexpected quality control mechanism for CCA synthesis | Escherichia coli |
kcat/KM [mM/s]
| kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 1.4 | - |
diphosphate | pH not specified in the publication, 37°C, diphosphorolysis reaction with substrate tRNA-A76 | Escherichia coli | |
| 3.8 | - |
diphosphate | pH not specified in the publication, 37°C, diphosphorolysis reaction with substrate tRNA-C75 | Escherichia coli | |
| 5.2 | - |
diphosphate | pH not specified in the publication, 37°C, diphosphorolysis reaction with substrate tRNA-C74 | Escherichia coli | |
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