. Author manuscript; available in PMC: 2012 Nov 15.

Published in final edited form as: Biochemistry. 2011 Oct 13;50(45):9724–9737. doi: 10.1021/bi2012788

Table 7.

Activities of the biotin carboxylase domain mutants for the HCO₃⁻-dependent ATPase reaction in the presence of free biotin^a.

	k_cat (min⁻¹)^b	% wild- type rate	K_{m MgATP} (mM)	k_cat/K_{m MgATP} (min⁻¹ mM⁻¹)	% wild-type k_cat/K_{m MgATP}
wild-type	0.93 ± 0.04	(100)	1.2 ± 0.1	0.75 ± 0.05	(100)
E218A	NA^c	---	---	---	---
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 ± 0.03^d	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/K1119Q^e	1.29 ± 0.02	140	0.257 ± 0.009	5.01 ± 0.08	670

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.

Data fitted to eqn 1 unless otherwise indicated.

NA = No activity detected.

Substrate inhibition with respect to MgATP data fitted to eqn 2, K_i = 2.8 ± 0.7 mM.

Values determined from global fit of biotin inhibition data to eqn 4.