Table 2.

Binding affinities and thermodynamic parameters of native protein and proteins with site-specific amino acid substitutions

ITC with a Microcal instrument was used for thermodynamic measurements, and data were analyzed using SEDPHAT (for Hd-SatA WT) and Origin software (for Hd-SatA mutants). NB, no binding.

Site no.	Hd-SatA protein	Ligand	Kd	ΔH	−TΔS
				kcal/mol	kcal/mol
1	WT	Neu5Ac	133 ± 22 nm	−2.21 ± 0.023	−7.172
		Neu5Gc	277 ± 58 nm	−2.986 ± 0.056	−5.96
2	S27N	Neu5Ac	5.52 ± 1.18 μm	2.458 ± 0.116	−9.625
		Neu5Gc	57.8 ± 19.3 μm	−0.635 ± 0.11	−5.155
3	S27K	Neu5Ac	24 ± 3.04 μm	−2.725 ± 0.126	−3.576
4	S362R	Neu5Ac	4.83 ± 0.44 μm	−1.107 ± 0.017	−6.138
5	S362A	Neu5Ac	418 ± 179 nm	−0.47 ± 0.0133	−8.224
6	S362K	Neu5Ac	5.37 ± 0.22 μm	−1.63 ± 0.03	−5.542
7	H333R	Neu5Ac	115 ± 52 nm	−2.850 ± 0.055	−6.615
8	H333A	Neu5Ac	31 ± 10 nm	−5.724 ± 0.052	−4.529
9	H333K	Neu5Ac	NB	NB	NB
10	R408A	Neu5Ac	NB	NB	NB
11	R408K	Neu5Ac	NB	NB	NB