. 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 6.

Activities of the biotin carboxylase domain mutants for the HCO₃⁻-dependent ATPase reaction^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	0.68 ± 0.03	(100)	0.89 ± 0.09	0.76 ± 0.05	(100)
E218A	NA^c	---	---	---	---
E218Q	0.034 ± 0.002	5	2.8 ± 0.4	0.012 ± 0.009	1.6
K245Q	1.0 ± 0.4	150	0.3 ± 0.2^d	3.06 ± 0.05	400
E305A	0.32 ± 0.01	48	0.82 ± 0.03	0.39 ± 0.02	50
E305Q	0.19 ± 0.02	30	0.98 ± 0.01	0.19 ± 0.08	25
E305D	0.55 ± 0.04	82	0.7 ± 0.1	0.79 ± 0.09	100
R301Q	0.13 ± 0.04	20	0.12 ± 0.02	1.1 ± 0.1	145
R301K	0.14 ± 0.03	20	0.053 ± 0.006^e	2.5 ± 0.5	330
R353K	0.33 ± 0.07	50	0.10 ± 0.01	3.2 ± 0.4	420
R353M	0.58 ± 0.01	85	0.30 ± 0.02	1.9 ± 0.1	250
E305A/K1119Q	0.336 ± 0.007	50	0.068 ± 0.007	4.9 ± 0.5	640
R353M/K1119Q	1.16 ± 0.06	170	0.22 ± 0.03	5.5 ± 0.6	720

Reaction conditions: 50 mM Bis-Tris, 25 mM Tricine, 25 mM glycine (pH 7.5), 25° C, 15 mM HCO₃⁻, 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 = 0.4 ± 0.2 mM.

Substrate inhibition, K_i = 4.1 ± 0.4 mM.