TABLE 1.

Michaelis-Menten kinetic parameters of WT and mutant AmpC enzymes for ceftolozane, ceftazidime, and nitrocefin^a

Enzyme, parameter	Mean value ± SE for indicated substrateb
	Ceftolozane	Ceftazidime	Nitrocefin
WT
kcat (s−1)	(1.04 ± 0.13) × 10−2	(4.85 ± 0.55) × 10−3	32.48 ± 3.2
Km (μM)	2,107.3 ± 341.2	353.5 ± 81.1	126.12 ± 23.63
kcat/Km (μM−1 s−1)	(4.91 ± 1.01) × 10−6	(1.37 ± 0.35) × 10−5	(2.58 ± 0.54) × 10−1
Fold changec	1	1	1
E247K
kcat (s−1)	1.48 ± 0.25	(6.58 ± 0.40) × 10−1	1.37 ± 0.18
Km (μM)	3,610.2 ± 704.3	1,016.9 ± 90.3	62.86 ± 17.67
kcat/Km (μM−1 s−1)	(4.11 ± 1.05) × 10−4	(6.47 ± 0.70) × 10−4	(2.18 ± 0.67) × 10−2
Fold change	83.7	47.2	0.085
G183D
kcat (s−1)	(1.80 ± 0.17) × 10−1	ND	1.25 ± 0.07
Km (μM)	1,174.3 ± 165.1	ND	73.53 ± 8.34
kcat/Km (μM−1 s−1)	(1.53 ± 0.26) × 10−4	(1.41 ± 0.02) × 10−4	(1.71 ± 0.22) × 10−2
Fold change	31.2	10.3	0.066
T96I
kcat (s−1)	NDd	ND	1.29 ± 0.05
Km (μM)	ND	ND	46.22 ± 4.51
kcat/Km (μM−1 s−1)	(1.99 ± 0.04) × 10−4	(2.27 ± 0.04) × 10−4	(2.78 ± 0.29) × 10−2
Fold change	40.5	16.6	0.107
ΔG229–E247
kcat (s−1)	ND	ND	1.00 ± 0.23
Km (μM)	ND	ND	222.66 ± 80.67
kcat/Km (μM−1 s−1)	(1.22 ± 0.02) × 10−4	(1.38 ± 0.02) × 10−4	(4.50 ± 1.93) × 10−3
Fold change	24.8	10.1	0.017

^aMichaelis-Menten kinetic parameters were obtained from fitting the respective data in Fig. S1, S2, and S3 in the supplemental material to the Michaelis-Menten equation.

^bExperiments were performed in triplicate; plus-minus values represent the standard errors reported from regression analysis.

^cRatio of enzyme specificity constant (k_cat/K_m) of mutant AmpC relative to that of WT AmpC.

^dND, not determined.