TABLE 1.

IC₅₀ and K_D values obtained from four different assays

IC₅₀ values (μm) were derived from competition studies with 100 μm chitohexaose in the DMAB assay, the pNP assay with pNP-GlcNAc₂, and the pNP assay with pNP-GlcNAc₃. Equilibrium dissociation constants (K_D, μm) derived from fluorescence quenching (Fig. 5) are shown. The K_D values for wild-type chitinase and the mutant W275G were derived from three-parameter fits of the corresponding ITC experiments assuming one set of sites (Fig. 6, panels A2, B2, C2, and D2, and Scheme 1). The values represent the means ± S.D. from at least three independent sets of the experiments. ND, not determined.

Inhibitor	WT				W275G
	IC50		KD		KD	IC50
	DMAB	pNP	Fluorescence	ITC	ITC	pNP
DEQ	3.9 ± 1.1	0.4 ± 0.1	0.2 ± 0.02	0.07	37	70 ± 28
IDA	6.4 ± 1.1	0.9 ± 0.2	0.4 ± 0.02	ND	ND	270 ± 33
SAN	7.6 ± 1.3	2.3 ± 0.3	0.6 ± 0.02	2.3	28	71 ± 26
CHE	11 ± 1.1	2.2 ± 0.01	0.7 ± 0.03	ND	ND	95 ± 34
IMI	21 ± 1.5	15 ± 3.2	1.0 ± 0.1	ND	ND	160 ± 78
PEN	59 ± 1.2	3.5 ± 0.5	4.0 ± 0.6	4.0	600	260 ± 21
PRO	83 ± 1.0	3.7 ± 0.9	4.7 ± 0.5	ND	ND	360 ± 42