Table 8.
Activities of the biotin carboxylase domain mutants for the ADP phosphorylation reaction using carbamoyl phosphate as the phosphoryl donora.
| kcat (min−1)b |
% wild- type rate |
Km CPc (mM) |
kcat/Km CP (min−1 mM−1) |
% wild-type kcat/Km CP |
|
|---|---|---|---|---|---|
| wild-type | 2.4 ± 0.1 | (100) | 2.6 ± 0.5 | 0.93 ± 0.02 | (100) |
| E218A | NAd | --- | --- | --- | --- |
| E218Q | 0.10 ± 0.01 | 4 | 1.5 ± 0.5 | 0.067 ± 0.006 | 7 |
| K245Qe | --- | --- | --- | --- | --- |
| E305A | 0.79 ± 0.02 | 33 | 0.31 ± 0.02 | 2.6 ± 0.1 | 280 |
| E305Q | 0.255 ± 0.006 | 10 | 3.2 ± 0.2 | 0.079 ± 0.002 | 8 |
| E305D | 5.4 ± 0.1 | 225 | 0.35 ± 0.05 | 16 ± 3 | 1720 |
| R301Q | 0.39 ± 0.06 | 16 | 0.89 ± 0.02 | 0.45 ± 0.02 | 50 |
| R301K | 0.108 ± 0.004 | 4.5 | 1.2 ± 0.3 | 0.092 ± 0.003 | 10 |
| R353K | 3.4 ± 0.1 | 140 | 5.3 ± 0.5 | 0.79 ± 0.05 | 85 |
| R353M | 24.9 ± 0.8 | 1000 | 0.9 ± 0.2 | 28 ± 4 | 3010 |
| E305A/K1119Q | 2.6 ± 0.2 | 100 | 1.4 ± 0.1 | 1.8 ± 0.1 | 190 |
| R353M/K1119Q | 0.30 ± 0.01 | 12 | 0.37 ± 0.09 | 0.8 ± 0.1 | 86 |
a
Reaction conditions: 50 mM Bis-Tris, 25 mM Tricine, 25 mM glycine (pH 7.5), 25° C, 7.5 mM MgCl2, 3.5 mM MgADP, carbamoyl phosphate (1.0–20 mM), 0.25 mM acetyl-CoA.
b
Data fitted to eqn 1 unless otherwise indicated.
c
CP = carbamoyl phosphate
d
NA = No activity detected.
e
Due to the extreme substrate inhibition with respect to carbamoyl phosphate reasonable values could not be determined.