Table 2.

Change of melting temperatures (ΔT_m) of recombinant wild-type and variant hGALE enzymes in the presence of various ligands.

ΔTm (K) Variant	Substrate UDP-Gal	UDP-Glc	UDP-GlcNAc	Cofactor NAD+	NADH
WT	−0.3 ± 1.2	−0.1 ± 0.5	2.2 ± 0.3***	1.1 ± 0.4**	0.5 ± 0.5
p.K161N	0.7 ± 0.5	− 0.4 ± 1.1	− 0.9 ± 0.3	0.6 ± 0.3	0.4 ± 0.4
p.D175N	3.0 ± 0.5***	3.0 ± 0.5***	6.5 ± 0.3***	5.5 ± 0.5***	4.2 ± 0.4***

ΔTm (K)	Substrate & Cofactor
Variant	NAD+and UDP-Gal	NAD+and UDP-Glc	NAD+and UDP-GlcNAc	NADH and UDP-Gal	NADH and UDP-Glc	NADH and UDP-GlcNAc
WT	3.4 ± 0.4***	3.4 ± 0.4***	6.7 ± 0.5***	8.3 ± 0.5***	8.3 ± 0.4***	12.0 ± 0.3***
p.K161N	−0.8 ± 1.9	−0.6 ± 0.6	−4.8 ± 0.9***	−0.6 ± 0.5	−0.3 ± 0.6	−1.1 ± 0.5
p.D175N	9.1 ± 0.6***	9.2 ± 1.9***	15.3 ± 0.5***	15.6 ± 0.4***	15.7 ± 0.6***	19.8 ± 0.4***

All ligands were added to a final concentration of 1 mM in 10 mM HEPES, pH 8.8. The change of melting temperature, ΔT_m, for mutant stability and ligand binding were calculated using equations (2) and (3) respectively.

$$\mathrm{\Delta }{\mathrm{T}}_{\mathrm{m}}=({\mathrm{T}}_{\mathrm{m}}\text{ of variant})-({\mathrm{T}}_{\mathrm{m}}\text{ of WT})$$ (2)

$$\mathrm{\Delta }{\mathrm{T}}_{\mathrm{m}}=({\mathrm{T}}_{\mathrm{m}}\text{ of protein with ligand})-({\mathrm{T}}_{\mathrm{m}}\text{ of protein without ligand})$$ (3)

T_mand ΔT_m values are the means ± SD; n = 3.

^**p< 0.001,

^***p < 0.0001 (One way ANOVA with Dunnett comparison test).