Table 7.
Activities of the biotin carboxylase domain mutants for the HCO3−-dependent ATPase reaction in the presence of free biotina.
| kcat (min−1)b |
% wild- type rate |
Km MgATP (mM) |
kcat/Km MgATP (min−1 mM−1) |
% wild-type kcat/Km MgATP |
|
|---|---|---|---|---|---|
| wild-type | 0.93 ± 0.04 | (100) | 1.2 ± 0.1 | 0.75 ± 0.05 | (100) |
| E218A | NAc | --- | --- | --- | --- |
| E218Q | 0.015 ± 0.005 | 2 | 1.0 ± 0.2 | 0.015 ± 0.006 | 2 |
| K245Q | 0.032 ± 0.005 | 3 | 1.81 ± 0.02 | 0.018 ± 0.04 | 2.5 |
| E305A | 0.41 ± 0.02 | 44 | 0.05 ± 0.01 | 8.2 ± 0.9 | 1090 |
| E305Q | 0.28 ± 0.03 | 30 | 1.7 ± 0.5 | 0.16 ± 0.01 | 21 |
| E305D | 0.88 ± 0.07 | 95 | 0.5 ± 0.1 | 1.7 ± 0.2 | 225 |
| R301Q | 0.11 ± 0.04 | 12 | 0.25 ± 0.03 | 0.47 ± 0.02 | 63 |
| R301K | 0.23 ± 0.01 | 25 | 0.98 ± 0.07 | 0.234 ± 0.02 | 31 |
| R353K | 0.62 ± 0.06 | 67 | 0.18 ± 0.03d | 3.4 ± 0.4 | 450 |
| R353M | 0.481 ± 0.006 | 52 | 0.050 ± 0.004 | 9.5 ± 0.6 | 1260 |
| E305A/K1119Q | 0.41 ± 0.02 | 45 | 0.21 ± 0.04 | 1.9 ± 0.3 | 250 |
| R353M/K1119Qe | 1.29 ± 0.02 | 140 | 0.257 ± 0.009 | 5.01 ± 0.08 | 670 |
a
Reaction conditions: 50 mM Bis-Tris, 25 mM Tricine, 25 mM glycine (pH 7.5), 25° C, 15 mM HCO3−, 10 mM biotin, 7.5 mM MgCl2, MgATP (0.09–3.0 mM), 0.25 mM acetyl-CoA.
b
Data fitted to eqn 1 unless otherwise indicated.
c
NA = No activity detected.
d
Substrate inhibition with respect to MgATP data fitted to eqn 2, Ki = 2.8 ± 0.7 mM.
e
Values determined from global fit of biotin inhibition data to eqn 4.