Table II.
Steady State Kinetic Parameters for PAP and Mutants
| Adenylyltransfer: | An + ATP ⇌ A(n+1) + PPi | ||||
|---|---|---|---|---|---|
| PAP Δ10 w.t.1 |
N189A
|
N226A
|
K215A
|
Y224F
|
|
| V1 (min−1) | 846 (± 127) | 665 (± 111) | 402 (± 81) | 191 (± 54) | 904 (± 511) |
| Kia (μM) | 93.2 (± 28.9) | 70.2 (± 16.1) | 54.0 (± 7.6) | 123.2 (± 17.9) | 92.8 (± 15.8) |
| Ka (μM) | 46.8 (± 12.4) | 106.0 (± 26.3) | 367.2 (± 87.0) | 195.0 (± 75.8) | 711.6 (± 448.3) |
| Kib (μM) | 71.5 (± 22.2) | 53.0 (± 9.8) | 36.6 (± 4.0) | 256.8 (± 45.7) | 121.1 (± 20.5) |
| Kb (μM) | 35.9 (± 12.6) | 80.0 (± 21.4) | 249.0 (± 62.3) | 406. (± 145.9) | 928.8 (± 583.3) |
|
| |||||
| Pyrophosphorolysis: | An + PPi⇌A(n−1) + ATP | ||||
|
| |||||
| V2 (min−1) | 189 (± 296) | 54.6 (± 53.4) | 21.9 (± 26.1) | n.d.2 | 3.8 (± 1.5) |
| K′a (μM) | 2270 (± 3600) | 1170 (± 1213) | 1678 (± 2089) | n.d | 121 (± 54) |
| Kip3(μM) | 28 (± 46) | 43 (± 42) | 18 (± 21) | 465 (± 62) | 285 (± 28) |
| Kp (μM) | 671 (± 1100) | 721 (± 726) | 563.0 (± 689) | n.d | 375 (± 167) |
|
| |||||
| Keq4 | 85 | 97 | 42 | n.d. | 95 |
|
| |||||
| Cytidylyltransfer: | An + CTP ⇌ An-C + PPi | ||||
|
| |||||
| V1 (min−1) | 8.6 (± 1.9) | 8.9 (± 1.8) | 1.04 (± 0.17) | 0.51 (± 0.24) | 4.7 (± 20.9) |
| Kia (μM) | 71.0 (± 18.1) | 62.4 (± 12.9) | 64.9 (± 11.9) | 216.7 (± 58.0) | 72.2 (± 17.0) |
| Ka (μM) | 64.2(± 22.5) | 145.6 (± 41.9) | 131.8 (± 32.8) | 129.9 (± 105.1) | 3.5 mM (± 15.9) |
| Kib (μM) | 114.6 (± 29.2) | 63.6 (± 12.8) | 69.2 (± 12.4) | 614.7 (± 265.8) | 96.3 (± 22.5) |
| Kb (μM) | 103.6 (± 36.2) | 148.3 (± 40.1) | 140.6 (± 33.1) | 368.5 (± 234.6) | 4.7 mM (± 21.3) |
|
| |||||
| Guanylyltransfer: | An + GTP ⇌ An-G + PPi | ||||
|
| |||||
| V1 (min−1) | 2.1 (± 0.6) | 0.64 (± 0.17) | |||
| Kia (μM) | 101.7 (± 34.1) | 119.4 (± 39.7) | |||
| Ka (μM) | 259.2 (± 90.0) | 246.6 (± 82.5) | |||
| Kib (μM) | 24.2 (± 4.8) | 26.7 (± 4.9) | |||
| Kb (μM) | 61.7 (± 27.8) | 54.9 (± 24.7) | |||
1
PAPΔ10 w.t. reactions (except GTP utilization) from reference (Balbo et al., 2005)
2
not determined
3
Kip values for K215, Y224F were determined from product inhibition experiments, these values were treated as fixed parameters in the analysis of the pyrophosphorolysis data using Equation 1, where the term K′aKip, which is equivalent to KiaKp, was used.
4
calculated from the Haldane equation (Gold et al., 1970), Keq = V1Kp/V2Kb