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. 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 2.

Activities of the biotin carboxylase domain mutants for the pyruvate carboxylation reactiona.

kcat
(min−1)b 		% wild-
type rate		 Km MgATP
(mM)		 kcat/Km MgATP
(min−1 mM−1)		 % wild-type
kcat/Km MgATP
wild-type 700 ± 80 (100) 0.27 ± 0.04 2590 ± 15 (100)
E218A NAc --- --- --- ---
E218Q 8.0 ± 0.3 1.1 0.03 ± 0.005d 270 ± 10 10
K245Q 71 ± 4 10 4.82 ± 0.07 15 ± 1 0.6
E305A 9.2 ± 0.2 1.3 0.09 ± 0.01 96 ± 12 4
E305Q 87 ± 1 12 1.32 ± 0.06 64 ± 1 2.5
E305D 187 ± 1 27 0.32 ± 0.08 580 ± 20 22
R301Q 18 ± 1 2.5 0.05 ± 0.02e 340 ± 10 13
R301K 3.8 ± 0.4 0.5 2.8 ± 0.2 1.4 ± 0.9 0.05
R353K 4.3 ± 0.1 0.6 0.12 ± 0.01 35 ± 4 1.4
R353M 28 ± 1 4 0.034 ± 0.009f 760 ± 10 30
E305A/K1119Q NA --- --- --- ---
R353M/K1119Q NA --- --- --- ---
a

Reaction conditions: 50 mM Bis-Tris, 25 mM Tricine, 25 mM glycine (pH 7.5), 25° C, 25 mM HCO3−, 12 mM pyruvate, 7.0 mM MgCl2, MgATP (0.075–3.0 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 = 1.1 ± 0.2 mM.

e

Substrate inhibition, Ki = 1.2 ± 0.3 mM.

f

Substrate inhibition, Ki = 1.8 ± 0.2 mM.