TABLE 2.

Enzymatic activity assays

	Assay	Km	kcat	kcat/Km
		μm	s−1	m−1 s−1
WT (1 mm Mg2+)	Cresol red	0.8 ± 0.2a	1.4 ± 0.1	1.8 × 106
	Enzcheck	≪5b	1.2 ± 0.2
Without Mg2+ (<0.08 μm)	Cresol red	6.0 ± 0.9	1.2 ± 0.2	0.2 × 106
WT with DTT	Cresol red	0.71 ± 0.05	1.6 ± 0.1	2.3 × 106
C4S	Cresol red	0.6 ± 0.2	1.7 ± 0.2	2.8 × 106
R268A	Enzcheck	8 ± 4	0.06 ± 0.01	0.008 × 106
F273A	Enzcheck	NMc	NM	NM
D131S	Enzcheck	5.2 ± 0.5	0.04 ± 0.01	0.008 × 106
D131E	Enzcheck	NM	NM	NM
D131N	Enzcheck	7 ± 2	0.04 ± 0.01	0.006 × 106
D131S/G78D	Enzcheck	9 ± 2	0.18 ± 0.02	0.02 × 106

^a Errors are based on 2–5 independent determinations of the kinetic constants and a minimal error of 10%.

^b The minimum concentrations (5 μm) of dUTP used in the test did not allow us to determine a value of K_m for the WT enzyme, confirming that the K_m is ≪5 μm.

^c NM, not measurable.