TABLE 1.

Kinetic and inhibition parameters of the CMY-2 variants^c

		CMY-2 variants
Substrate	Kinetics parameters	Without mutation (CMY-2)	V211S (CMY-42)	N346Y	A114E_N346Y	Q120K_N346Y	V211S_N346Y	A114E_Q120K_ V211S_N346Y (CMY-185)
Nitrocefin
	kcat (s−1)	2.4 × 102 ± 0.5 × 102	3.2 × 102 ± 1.02 × 102	61 ± 5	57 ± 10	1.2 × 102 ± 0.9 × 102	85 ± 18	17 ± 9
	Km (µM)	17 ± 5	29 ± 12	25 ± 3	16 ± 4	50 ± 40	35 ± 10	39 ± 24
	Ki (µM)	1.6 × 102 ± 1.0 × 102	1.3 × 102 ± 0.7 × 102	87 ± 25	68 ± 37	14 ± 12	1.0 × 102 ± 0.5 × 102	18 ± 13
	kcat/Km (µM−1 s−1)	14 ± 5	11 ± 5	2.5 ± 0.4	3.6 ± 1.2	2.4 ± 2.5	2.4 ± 0.9	0.4 ± 0.4
Cephalothin
	kcat (s−1)	54 ± 0.8	83 ± 7	18 ± 7	3.3 ± 0.1	42 ± 5	9.6 ± 0.2	14 ± 1
	Km (µM)	4.1 ± 0.3	7.5 ± 1.1	3.9 ± 0.1	1.4 ± 0.1	18 ± 3	4.4 ± 0.3	19 ± 1
	kcat/Km (µM−1 s−1)	13 ± 1	11 ± 2	4.7 ± 1.8	2.3 ± 0.2	2.4 ± 0.5	2.2 ± 0.2	0.8 ± 0.1
Ceftazidime
	kcat (s−1)	N.D.	1.5 ± 0.2	N.D.	N.D.	7.7 × 10−2 ± 0.3 × 10−2	0.3 ± 0.0	0.1 ± 0.0
	Km (µM)	N.M.	74 ± 10	N.M.	N.M.	5.3 ± 0.5	37 ± 2	2.2 ± 0.3
	Ki app (µM)	0.7 ± 0.1	N.M.	0.7 ± 0.2	0.3 ± 0.0	N.M.	N.M.	N.M.
	kcat/Km (µM−1 s−1)	N.C.	2.0 × 10−2 ± 0.3 × 10−2	N.C.	N.C.	1.5 × 10−2 ± 0.2 × 10−2	9.0 × 10−3 ± 0.7 × 10−3	5.3 × 10−2 ± 0.7 × 10−2
Cefiderocol
	kcat (s−1)	N.D.	3.4 × 10−2 ± 0.2 × 10−2	N.D.	N.D.	N.D.	4.9 × 10−2 ± 0.3 × 10−2	6.9 × 10−3 ± 0.4 ×10−3 a
	Km (µM)	N.M.	68 ± 11	N.M.	N.M.	N.M.	6.9 ± 1.5	N.M.
	Ki app (µM)	8.0 ± 1.3	N.M.	0.7 ± 0.0	0.4 ± 0.0	0.4 ± 0.0	N.M.	0.2 ± 0.0
	kcat/Km (µM−1 s−1)	N.C.	4.9 × 10−4 ± 0.9 × 10−4	N.C.	N.C.	N.C.	7.1 × 10−3 ± 1.6 × 10−3	3.0 × 10−2 ± 0.2 × 10−2 b
Avibactam
	Ki app (µM)	6.3 × 10−2 ± 1.2 × 10−2	3.4 × 10−2 ± 0.6 × 10−2	41 ± 6	80 ± 9	5.9 × 102 ± 0.2 × 102	14 ± 4	1.3 × 103 ± 0.1 × 103
	k2/K (M−1 s−1)	2.8 × 104 ± 0.1 × 104	2.3 × 104 ± 0.1 × 104	59 ± 6	41 ± 0.4	32 ± 0.4	66 ± 6	7.4 ± 0.3
	k−2 (s−1)	1.2 × 10−4 ± 0.2 × 10−4	1.5 × 10−4 ± 0.2 × 10−4	1 × 10−5 ± 16 × 10−5	1.1 × 10−4 ± 0.0 × 10−4	2.0 × 10−3 ± 0.1 × 10−3	4.4 × 10−4 ± 2.1 × 10−4	7.0 × 10−4 ± 0.5 × 10−4
	koff (s−1)	7.3 × 10−5 ± 0.0 × 10−5	6.0 × 10−5 ± 0.0 × 10−5	2.6 × 10−6 ± 0.0 × 10−6	9.0 × 10−7 ± 0.0 × 10−7	4.0 × 10−6 ± 0.0 ×10−6	2.4 × 10−6 ± 0.0 × 10−6	4.8 × 10−7 ± 0.0 × 10−7

^a Due to the small K_m value of CMY-185 for cefiderocol, the k_cat value was determined with the measured velocities at the steady state (cefiderocol concentration range: 3.7–59.4 µM) and the K_{i app} value assumed to K_m.

^b The k_cat/K_{i app} value is shown.

^c The values are the mean ± standard error of three independent measurements. N.M. = not measured; N.D. = not detected; N.C. = not calculated.