Table 1.

Steady-state kinetic analyses of wildtype OXA-48 and variants with nitrocefin, meropenem, and imipenem, as determined spectrophotometrically. Assays were performed using nitrocefin (5–1500 μm) and enzyme (25–500 pm) in 50 mm sodium phosphate, pH 7.5, or meropenem (1.75–500 μm), or imipenem (5–500 μm) with enzyme (1.5–400 nm) in 50 mm sodium phosphate, pH 7.5, supplemented with 50 mm sodium bicarbonate. Kinetic parameters are means ± standard deviations (n = 3)

OXA-48	Substrate	kcat	Km	kcat/Km
		s−1	μm	(m−1s−1) × 10−6
WT	Nitrocefin	593.2 ± 18.3	23.9 ± 3.5	24.8 ± 3.7
	Meropenem	0.087 ± 0.01	<1.9a	>0.045a
	Imipenem	11.4 ± 0.5	57.7 ± 9.3	0.20 ± 0.03
V120L	Nitrocefin	287.1 ± 11.7	184.1 ± 22.8	1.5 ± 0.20
	Meropenem	1.1 ± 0.08	51.0 ± 11.8	0.021 ± 0.005
	Imipenem	0.49 ± 0.006	6.8 ± 0.6	0.071 ± 0.006
V120I	Nitrocefin	323.2 ± 11.1	40.1 ± 4.9	8.1 ± 1.02
	Meropenem	0.94 ± 0.01	12.6 ± 1.4	0.075 ± 0.008
	Imipenem	17.9 ± 1.4	208.2 ± 45.2	0.086 ± 0.00
W105F	Nitrocefin	178.1 ± 16.7	298.4 ± 37.8	0.60 ± 0.09
	Meropenem	0.038 ± 0.0007	<1.75a	>0.022a
W105A	Nitrocefin	70.72 ± 1.5	218.1 ± 9.5	0.32 ± 0.02
	Meropenemb
L158V	Nitrocefin	54.9 ± 4.3	16.1 ± 3.3	3.4 ± 0.8
	Meropenem	0.0081 ± 0.0002	<2a	>0.0041a
L158I	Nitrocefin	59.8 ± 3.5	8.1 ± 2.2	7.4 ± 2.1
	Meropenem	0.031 ± 0.005	7.1 ± 4.1	0.0044 ± 0.003

^aThe K_m values shown are the lowest [S] at which turnover could be accurately measured. The actual K_m values are thus lower and the k_cat/K_m values are larger than those given.

^bNot determined because of slow reaction rate.