FIGURE 4.
Inhibition of NgaP activity by GalNAc thiazoline and GlcNAc thiazoline. A, aliquots of 50 nmol of pNP-β-GalNAc were incubated with 17 nm enzyme at 37 °C for 30 min in 100 μl of 25 mm acetate buffer (pH 6.0). The concentrations of GalNAc thiazoline used were 2.5, 5, and 10 nm (white bars), and those of GlcNAc thiazoline used were 100, 200, and 400 μm (gray bars). The type of inhibition was determined using the Henderson plot (B) and Lineweaver-Burk plot (C) with GalNAc thiazoline and GlcNAc thiazoline, respectively. The reaction mixture containing different amounts of pNP-β-GalNAc and 17 nm enzyme was incubated at 37 °C for 30 min in 100 μl of 25 mm acetate buffer (pH 6.0). B, the concentrations of GalNAc thiazoline used were 5∼40 nm, and those of pNP-β-GalNAc used were 0.25 mm (○), 0.5 mm (●), and 0.75 mm (□). C, the concentrations of GlcNAc thiazoline used were 800 μm (●), 400 μm (○), 200 μm (■), 100 μm (□), and 0 μm (▴). The Ki values of NgaP for GalNAc thiazoline (D) and GlcNAc thiazoline (E) were determined by the Morrison equation (18) and Dixon plots (19), respectively. D, aliquots of 50 nmol of pNP-β-GalNAc were incubated with 17 nm enzyme at 37 °C for 30 min in 100 μl of 25 mm acetate buffer (pH 6.0). The Vi/V0 ratio was plotted against inhibitor concentration. E, the concentrations of pNP-β-GalNAc used were 2 mm (●), 1 mm (○), 0.5 mm (■), 0.25 mm (□), and 0.167 mm (▴). The reaction mixture containing different amounts of pNP-β-GalNAc, 17 nm enzyme, and different amounts of inhibitor in 100 μl of 25 mm acetate buffer (pH 6.0) was incubated at 37 °C for 30 min. Values are means of triplicate determinations.