Cloned (Comment) | Organism |
---|---|
expression in Escherichia coli | Bos taurus |
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 | 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 | 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 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 | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | optimal concentration is about 5 mM | Bos taurus |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Bos taurus | - |
bovine PAP | - |
Purification (Comment) | Organism |
---|---|
- |
Bos taurus |
Storage Stability | Organism |
---|---|
-80°C, 10-20 mg protein/ml, flash frozen | Bos taurus |
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 | Comment | Organism |
---|---|---|
PAP | - |
Bos taurus |
poly(A) polymerase | - |
Bos taurus |
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 |