Table 2.

Apparent kinetic constants for wild-type and mutant enzymes.

Protein	Substrate	kcat (s−1)	KM (mM)	kcat/KM (M−1 s−1)	kcat/KM HAa (M−1 s−1)
WTb	Citrate	0.35 ± 0.01	0.18 ± 0.03	1.94 × 103
WT	Citrate	0.69 ± 0.01	0.54 ± 0.04	1.29 × 103
H147F	Citrate	(1.3 ± 0.1) × 10−2	10.1 ± 1.3	1.32
T284V	Citrate	(1.2 ± 0.1) × 10−4	5.72 ± 1.47	2.02 × 10−2
R288K	Citrate	(2.2 ± 0.1) × 10−2	3.76 ± 0.71	5.90
R288A	Citrate	(7.53 ± 0.27) × 10−4	0.41 ± 0.07	1.86
H425A	Citrate	(1.48 ± 0.09) × 10−3	15.7 ± 1.8	9.41 × 10−2
H425N	Citrate	(7.67 ± 0.10) × 10−2	0.89 ± 0.05	8.63 × 101
N542L	Citrate	0.79 ± 0.07	0.71 ± 0.18	1.12 × 103
WTb	ATP	0.43 ± 0.02	0.13 ± 0.03	3.33 × 103
WT	ATP	0.35 ± 0.01	0.05 ± 0.01	6.64 × 103
M274W	ATP	0.63 ± 0.04	0.15 ± 0.04	4.36 × 103
H425N	ATP	0.13 ± 0.01	0.19 ± 0.02	6.77 × 102
N428L	ATP	(3.90 ± 0.06) × 10−3	0.17 ± 0.01	2.28 × 101
WT	ahLys	0.85 ± 0.02	0.79 ± 0.02	1.08 × 103	21
R297A	ahLys	2.83 ± 0.30	3.18 ± 0.63	8.91 × 102
L423E	ahLys	(6.00 ± 0.15) × 10−2	2.74 ± 1.36	2.19 × 101	142
Q447A	ahLys	1.29 ± 0.30	3.64 ± 1.66	3.55 × 102	19
F471Y	ahLys	1.68 ± 0.34	1.58 ± 0.73	1.06 × 103
Y479A	ahLys	4.93 ± 1.91	> 20	< 200
Y479R	ahLys	0.46 ± 0.11	5.77 ± 2.01	7.94 × 101	38
Y483A	ahLys	0.18 ± 0.06	5.12 ± 2.18	3.58 × 101
Y483F	ahLys	1.70 ± 0.65	16.5 ± 7.9	1.03 × 102
Y483C	ahLys	(7.50 ± 1.67) × 10−2	3.54 ± 1.38	2.12 × 101	24
N551Ac	ahLys	--			23
E552A	ahLys	(1.21 ± 0.10) × 10−3	> 50	< 2	4
N553Ac	ahLys	--			22

^aApparent kinetic constants with surrogate nucleophile were determined for mutant enzymes with large effect on ahLys; for residues with multiple designed mutations, only a single mutant was tested.

^bValues from Bailey et al. (19).

^cNo activity was detected for N551A and N553A with 30 μM enzyme concentration.