TABLE 2.

Steady-state substrate utilization by mutant CARDO enzymes^a

Oxy of CARDO	Substrate	Km (μM)	kcat (10−2 s−1)	kcat/Km (10−4 M−1 s−1)	O2 uptake (nmol s−1)c	Substrate consumption (nmol s−1)	Substrate/O2b
WT	CAR	NDd	ND	ND	0.840 ± 0.002	0.651 ± 0.089	0.78
WT	FN	ND	ND	ND	0.361 ± 0.026	0.195 ± 0.071	0.54
I262V derivative	CAR	50.7 ± 12.3	4.62 ± 0.53	9.10 ± 1.28	0.250 ± 0.015	0.146 ± 0.074	0.58
F275W derivative	FN	ND	ND	ND	0.507 ± 0.030	0.264 ± 0.035	0.52
Q282N derivative	CAR	54.6 ± 4.4	4.00 ± 0.12	7.33 ± 0.41	0.310 ± 0.018	0.128 ± 0.022	0.41
Q282Y derivative	CAR	54.8 ± 21.1	5.49 ± 0.02	10.0 ± 2.2	0.880 ± 0.024	0.564 ± 0.064	0.64

^aThe indicated values represent means and standard deviations obtained from three determinations. O₂ consumption was corrected for the background activity observed in the absence of substrate.

^bEach value is the ratio of the rate of substrate consumption to the average rate of O₂ consumption over the duration of the assay.

^cO₂ concentrations without a substrate were measured by using catalase during the reaction as a negative control, and the oxygen concentration did not increase under any conditions.

^dND, not determined.