Table 6.

Impact of APH(3′)-IIIa mutations on resistance, enzyme-drug binding affinity, and catalysis

APH(3′)-IIIa	MIC (μM)a	Ka (M−1)b	Km (μM)c	kcat (s−1)c
Neo
none	6	n.a.	n.a.	n.a.
wt	1600	(4.2 ± 1.5) × 107	18.2 ± 1.6	1.7 ± 0.1
E157A	800	(7.6 ± 0.2) × 106	14.7 ± 1.7	1.3 ± 0.1
E160A	1600	(1.4 ± 0.1) × 106	25.2 ± 5.1	2.4 ± 0.2
D190A	6	(1.9 ± 0.2) × 106	n.d.	n.d.
D193A	200	(1.9 ± 0.4) × 107	5.9 ± 1.0	0.7 ± 0.1
E230A	1600	(9.1 ± 3.4) × 106	10.0 ± 1.0	1.3 ± 0.1
D261A	12	(1.3 ± 0.3) × 105	3.3 ± 1.5	0.02 ± 0.01
E262A	6	(2.1 ± 0.1) × 104	21.2 ± 5.1	1.9 ± 0.1
ΔF264	12	(1.4 ± 0.1) × 104	10.7 ± 1.8	0.9 ± 0.1
KanB
none	6	n.a.	n.a.	n.a.
wt	1600	(1.7 ± 0.2) × 107	19.3 ± 6.8	1.9 ± 0.2
E157A	800	(2.2 ± 0.9) × 107	44.1 ± 5.7	2.5 ± 0.1
E160A	400	(2.1 ± 0.1) × 105	42.9 ± 7.2	2.7 ± 0.2
D190A	6	(1.8 ± 0.1) × 106	n.d.	n.d.
D193A	1600	(1.3 ± 0.1) × 107	38.7 ± 9.1	2.7 ± 0.2
E230A	1600	(1.1 ± 0.1) × 107	39.8 ± 4.5	2.1 ± 0.1
D261A	12	(1.4 ± 0.1) × 105	9.1 ± 1.0	0.2 ± 0.01
E262A	6	(2.2 ± 0.6) × 103	36.4 ± 4.6	1.2 ± 0.1
ΔF264	12	(6.3 ± 0.2) × 103	16.4 ± 3.0	0.9 ± 0.1

^aAntibacterial activities were assayed in at least two independent experiments. For each drug, the MIC (minimal inhibitory concentration) value obtained was identical in each of the experiments.

^bValues of K_a were derived from fits of the ITC profiles shown in Figure 7. The indicated uncertainties reflect the standard deviations of the experimental data from the fitted curves. n.a. denotes not applicable.

^cValues of K_m and k_cat were derived from the fits of v versus [S] plots as described in the Materials and Methods, with the indicated uncertainties reflecting the standard deviations of the experimental data from the fitted curves. n.d. denotes not detectable.