Mechanistic studies on human N-acetylgalactosamine kinase

Figures & data

Figure 1.  (a) The reaction catalyzed by N-acetylgalactosamine kinase. (b) The active site of GALK2. Only the substrates and key residues are shown. The loop comprising Thr-184 to Gly-186 accommodates the N-acetyl group of N-acetylgalactosamine (GalNAc). In galactokinase, the threonine and glycine residues in this loop are substituted for bulkier methionine and cysteine side chains respectively. Asp-190 is located with the carboxyl group in between the C1-OH of the sugar and the γ-phosphate of the nucleotide. This residue is likely to be important in the catalytic mechanism of the enzyme and its ionization state is probably influenced by Arg-43. The figure was drawn using PyMol (www.pymol.org) and the PDB file 2A2D⁸. (c) Structures of N-acetylgalactosamine kinase inhibitors used in this study.

Figure 2.  Purification of recombinant human GALK2. Samples taken during the purification procedure (see methods) were analyzed by 10% SDS-PAGE. The sizes of molecular mass markers (M) are shown on the left. U, extract from cells prior to induction; I, extracts from induced cells prior to harvesting by centrifugation; S, protein solution extracted from cells by sonication and clarified by centrifugation; F, material which passed through the nickel–agarose column; E, protein eluted from this column in the presence of 250 mM imidazole.

Figure 3.  Recombinant human GALK2 (50 nM) is an active N-acetylgalactosamine kinase. In (a), the concentration of ATP was maintained at a constant level of 0.5 mM and N-acetylgalactosamine (GalNAc) was varied. The resulting data were fit (i) to the Michaelis–Menten equation (giving values of K_m,GalNAc = 40 ± 14 μM and k_cat = 1.0 ± 0.1 s⁻¹) and (ii) to a modified version of this equation which accounts for substrate inhibition (giving values of K_is = 13 ± 8 mM, K_m = 73 ± 27 mM, and k_cat = 1.2 ± 0.1 s⁻¹). In (b) the GalNAc concentration was maintained at 5 mM and the concentration of ATP was varied (giving values of K_m,ATP = 14 ± 3 μM and k_cat = 1.0 ± 0.1 s⁻¹). Each point represents the mean of two or three separate determinations and the error bars the standard deviations of these means.

Figure 4.  The inhibition of GALK2 (50 nM) by α-methylgalactopyranoside. Inhibition was studied over a range of α-methylgalactopyranoside concentrations and three different ATP concentrations: 8 μM (inverted triangles, ▾), 15 μM (squares, ▪), and 60 μM (upright triangles, ▴). (a) In a Dixon plot the lines do not converge, suggesting that the inhibition is not competitive. (b) The converging lines on a plot of [ATP]/rate against [inhibitor] suggest that the mode of inhibition is uncompetitive with respect to ATP. In all cases the N-acetylgalactosamine concentration was 1.0 mM.

Figure 5.  The effects of pH on the turnover number, k_cat. The GALK2 concentration was 230 nM in these experiments. The line represents a fit to the equation k_cat = k_cat,lim(1 + [H⁺]/K_a1 + K_a2/[H⁺]).

Thoden JB, Holden HM. The molecular architecture of human N-acetylgalactosamine kinase. J Biol Chem 2005;280:32784–91.

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