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Literature summary for 2.7.7.19 extracted from
- Biochemical and structural insights into substrate binding and catalytic mechanism of mammalian poly(A) polymerase (2004), J. Mol. Biol., 341, 911-925.
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| expression in Escherichia coli | Bos taurus |
Crystallization (Commentary)
| Crystallization (Comment) | Organism |
|---|---|
| Crystals of PAP complexed to 3'-dATP and Mn2+ are grown from a solution made of 22% (w/v) polyethylene glycol 8000, 100 mM Mes (pH 6), 120 mM ammonium sulfate, 5 mM CaCl2, 2 mM MnCl2, and 2 mM beta-mercaptoethanol. Cocrystallization of PAP with 3'-dATP and MgCl2 is performed under conditions similar to those reported, with the exception that 5 mM MgCl2 is used instead of 2 mM MnCl2. Crystals are of the space group p212121, with one monomer per asymmetric unit and a solvent content of ~55%. | Bos taurus |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| D167N | strong reduction of catalytic efficiency | Bos taurus |
| D167N/N202A | sharp decrease in catalytic efficiency | Bos taurus |
| D167N/T317G | sharp decrease in catalytic efficiency | Bos taurus |
| D167N/V247R | sharp decrease in catalytic efficiency | Bos taurus |
| F100D | strong reduction of catalytic efficiency | Bos taurus |
| G203H | strong reduction of catalytic efficiency | Bos taurus |
| K228A | increase in Km for ATP | Bos taurus |
| K232A | increase in Km for ATP | Bos taurus |
| N202A | high Km value for RNA, strong reduction of catalytic efficiency, incorporates etheno-ATP and N1-methyl-ATP better than wild-type, but incorporates N6-methyl-ATP poorly | Bos taurus |
| N239A | kcat for ATP is reduced fourfold | Bos taurus |
| R199A | reduces kcat for RNA about 30-fold | Bos taurus |
| T317G | increase in Km for ATP, tolerates etheno-ATP and GTP, rejects N6-methyl-ATP | Bos taurus |
| V156A | strong reduction of catalytic efficiency | Bos taurus |
| V247R | kcat for ATP is reduced 2000-fold, strong reduction of catalytic efficiency | Bos taurus |
| Y237A | increase in Km for ATP | Bos taurus |
Inhibitors
| Inhibitors | Comment | Organism | Structure |
|---|---|---|---|
| CaCl2 | remaining activity 3% | Bos taurus |  |
| CoCl2 | remaining activity 75% | Bos taurus | |
| CuCl2 | remaining activity 0.1% | Bos taurus | |
| Mn2+ | high concentrations | Bos taurus | |
| Zn2+ | inhibits PAP activity at concentrations above 10 µM in the presence of 5 mM MgCl2 | Bos taurus | |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.136 | - |
ATP | mutant D167N | Bos taurus | |
| 0.154 | - |
ATP | mutant D167N/N202A | Bos taurus | |
| 0.207 | - |
ATP | mutant N239A | Bos taurus | |
| 0.213 | - |
ATP | mutant F153A | Bos taurus | |
| 0.229 | - |
ATP | wild-type, reactions are started with the addition of PAP and incubated at 37 °C for 15 minutes. Reactions are stopped by the addition of 8 microL of stop buffer (formamide with trace amount of bromophenol blue dye) | Bos taurus | |
| 0.232 | - |
ATP | mutant V156A | Bos taurus | |
| 0.247 | - |
ATP | mutant A319R | Bos taurus | |
| 0.248 | - |
ATP | mutant K158A | Bos taurus | |
| 0.255 | - |
ATP | mutant R199A | Bos taurus | |
| 0.256 | - |
ATP | mutant V154A | Bos taurus | |
| 0.266 | - |
ATP | mutant V247A | Bos taurus | |
| 0.275 | - |
ATP | mutant N202A | Bos taurus | |
| 0.297 | - |
ATP | mutant V247R | Bos taurus | |
| 0.298 | - |
ATP | mutant Q323A | Bos taurus | |
| 0.357 | - |
ATP | mutant V206A | Bos taurus | |
| 0.361 | - |
ATP | mutant F100D | Bos taurus | |
| 0.503 | - |
ATP | mutant K232A | Bos taurus | |
| 0.51 | - |
RNA (A)15 | mutant A319R | Bos taurus | |
| 0.627 | - |
ATP | mutant D167N/T317G | Bos taurus | |
| 0.74 | - |
RNA (A)15 | mutant K232A | Bos taurus | |
| 1.002 | - |
ATP | mutant T317G | Bos taurus | |
| 1.34 | - |
RNA (A)15 | wild-type | Bos taurus | |
| 1.402 | - |
ATP | mutant Y237A | Bos taurus | |
| 1.43 | - |
RNA (A)15 | mutant K228A | Bos taurus | |
| 1.47 | - |
RNA (A)15 | mutant V154A | Bos taurus | |
| 1.88 | - |
RNA (A)15 | mutant Q323A | Bos taurus | |
| 2.191 | - |
ATP | mutant K228A | Bos taurus | |
| 2.63 | - |
RNA (A)15 | mutant Y237A | Bos taurus | |
| 2.8 | - |
RNA (A)15 | mutant V206A | Bos taurus | |
| 3.13 | - |
RNA (A)15 | mutant T317G | Bos taurus | |
| 5.31 | - |
RNA (A)15 | mutant K158A | Bos taurus | |
| 6.94 | - |
RNA (A)15 | mutant N239A | Bos taurus | |
| 7.32 | - |
RNA (A)15 | mutant V156A | Bos taurus | |
| 9.85 | - |
RNA (A)15 | mutant V247A | Bos taurus | |
| 10.21 | - |
RNA (A)15 | mutant R199A | Bos taurus | |
| 11.16 | - |
RNA (A)15 | mutant V247R | Bos taurus | |
| 11.44 | - |
RNA (A)15 | mutant N202A | Bos taurus | |
| 12.72 | - |
RNA (A)15 | mutant G203H | Bos taurus | |
| 13.15 | - |
RNA (A)15 | mutant D167N | Bos taurus | |
| 14.37 | - |
RNA (A)15 | mutant F100D | Bos taurus | |
| 16.41 | - |
RNA (A)15 | mutant D167N/N202A | Bos taurus | |
| 18.82 | - |
RNA (A)15 | mutant D167N/T317G | Bos taurus | |
| 22.84 | - |
RNA (A)15 | mutant D167N/V247R | Bos taurus | |
| 24.99 | - |
RNA (A)15 | mutant F153A | Bos taurus |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Mg2+ | optimal concentration is about 5 mM | Bos taurus | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Bos taurus | - |
bovine PAP | - |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
- |
Bos taurus |
Storage Stability
| Storage Stability | Organism |
|---|---|
| -80°C, 10-20 mg protein/ml, flash frozen | Bos taurus |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| ATP + RNA (A)15 | - |
Bos taurus | diphosphate + RNA (A)16 | - |
? | |
| ATP + RNA(A)15 | - |
Bos taurus | diphosphate + RNA(A)16 | - |
? | |
| ATP + RNAn | adenine can bind in two different configurations in the PAP active site | Bos taurus | diphosphate + RNAn+1 | - |
? | |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| PAP | - |
Bos taurus |
| poly(A) polymerase | - |
Bos taurus |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.002 | - |
ATP | mutant V247R | Bos taurus | |
| 0.0025 | - |
RNA(A)15 | mutant D167N/V247R | Bos taurus | |
| 0.006 | - |
ATP | mutant D167N/N202A | Bos taurus | |
| 0.006 | - |
RNA(A)15 | mutant G203H | Bos taurus | |
| 0.007 | - |
ATP | mutant D167N/T317G | Bos taurus | |
| 0.009 | - |
ATP | mutant D167N | Bos taurus | |
| 0.015 | - |
ATP | mutant F100D | Bos taurus | |
| 0.018 | - |
ATP | mutant V156A | Bos taurus | |
| 0.022 | - |
ATP | mutant N202A | Bos taurus | |
| 0.08 | - |
RNA(A)15 | mutant N202A | Bos taurus | |
| 0.098 | - |
ATP | mutant R199A | Bos taurus | |
| 0.1 | - |
RNA(A)15 | mutant D167N/N202A | Bos taurus | |
| 0.18 | - |
RNA(A)15 | mutant V247R | Bos taurus | |
| 0.26 | - |
RNA(A)15 | mutant D167N/T317G | Bos taurus | |
| 0.308 | - |
ATP | mutant F153A | Bos taurus | |
| 0.39 | - |
RNA(A)15 | mutant V156A | Bos taurus | |
| 0.67 | - |
RNA(A)15 | mutant F100D | Bos taurus | |
| 0.82 | - |
ATP | mutant K158A | Bos taurus | |
| 0.82 | - |
RNA(A)15 | mutant R199A | Bos taurus | |
| 1.055 | - |
ATP | mutant N239A | Bos taurus | |
| 1.073 | - |
ATP | mutant Y237A | Bos taurus | |
| 1.15 | - |
ATP | mutant K228A | Bos taurus | |
| 1.24 | - |
ATP | mutant V154A | Bos taurus | |
| 1.25 | - |
ATP | mutant T317G | Bos taurus | |
| 1.411 | - |
ATP | mutant V247A | Bos taurus | |
| 1.43 | - |
RNA(A)15 | mutant D167N | Bos taurus | |
| 1.476 | - |
ATP | mutant K232A | Bos taurus | |
| 3.19 | - |
ATP | mutant V206A | Bos taurus | |
| 4.203 | - |
ATP | wild-type, reactions are started with the addition of PAP and incubated at 37 °C for 15 minutes. Reactions are stopped by the addition of 8 microL of stop buffer (formamide with trace amount of bromophenol blue dye) | Bos taurus | |
| 4.88 | - |
ATP | mutant Q323A | Bos taurus | |
| 6.092 | - |
ATP | mutant A319R | Bos taurus | |
| 6.76 | - |
RNA(A)15 | mutant K228A | Bos taurus | |
| 6.86 | - |
RNA(A)15 | mutant T317G | Bos taurus | |
| 8.18 | - |
RNA(A)15 | mutant K158A | Bos taurus | |
| 10.52 | - |
RNA(A)15 | mutant F153A | Bos taurus | |
| 11.37 | - |
RNA(A)15 | mutant N239A | Bos taurus | |
| 12.58 | - |
RNA(A)15 | mutant V206A | Bos taurus | |
| 19.25 | - |
RNA(A)15 | mutant Y237A | Bos taurus | |
| 19.71 | - |
RNA(A)15 | mutant Q323A | Bos taurus | |
| 20.34 | - |
RNA(A)15 | mutant K232A | Bos taurus | |
| 22.75 | - |
RNA(A)15 | mutant A319R | Bos taurus | |
| 22.78 | - |
RNA(A)15 | wild-type | Bos taurus | |
| 28.96 | - |
RNA(A)15 | mutant V154A | Bos taurus | |
| 34.5 | - |
RNA(A)15 | mutant V247A | Bos taurus |
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