Publications

• Identification of the GalNAc kinase amino acid sequence.

  Pastuszak I., O'Donnell J., Elbein A.D.

  A new kinase that forms GalNAc-1-P was purified from pig kidney cytosol and identified on gels by labeling with N3-[32P]ATP (Pastuszak, I., Drake, R., and Elbein, A. D. (1996) J. Biol. Chem. 271, in press). A 50-kDa labeled protein was eluted, digested with trypsin, and the sequences of four pepti ... >> More

  A new kinase that forms GalNAc-1-P was purified from pig kidney cytosol and identified on gels by labeling with N3-[32P]ATP (Pastuszak, I., Drake, R., and Elbein, A. D. (1996) J. Biol. Chem. 271, in press). A 50-kDa labeled protein was eluted, digested with trypsin, and the sequences of four peptides representing 49 amino acids showed 90% identity to sequence of human galactokinase reported to be on chromosome 15. To resolve this dilemma, activities and substrate specificities of galactokinase and GalNAc kinase from human and pig kidney, as well as of galactokinase from the yeast clone transfected with the cDNA from presumptive human galactokinase, were compared. The purified galactokinases phosphorylated galactose, but not GalNAc, whereas GalNAc kinase also phosphorylated galactose when this sugar was present at millimolar concentrations. Extracts of gal 1(-) yeast clone, transfected with presumptive human galactokinase cDNA, had very low galactokinase activity even when yeast were grown on galactose, but good activity with GalNAc. On the other hand, the wild type yeast phosphorylated galactose, but not GalNAc. These data indicate that the sequence reported for galactokinase on chromosome 15 is that of GalNAc kinase, which can phosphorylate galactose when this sugar is present at millimolar concentrations. This transfection thus allows the yeast mutant to grow slowly on galactose-containing media. << Less

  J. Biol. Chem. 271:23653-23656(1996) [PubMed] [EuropePMC]

• The molecular architecture of human N-acetylgalactosamine kinase.

  Thoden J.B., Holden H.M.

  Galactokinase plays a key role in normal galactose metabolism by catalyzing the conversion of alpha-d-galactose to galactose 1-phosphate. Within recent years, the three-dimensional structures of human galactokinase and two bacterial forms of the enzyme have been determined. Originally, the gene en ... >> More

  Galactokinase plays a key role in normal galactose metabolism by catalyzing the conversion of alpha-d-galactose to galactose 1-phosphate. Within recent years, the three-dimensional structures of human galactokinase and two bacterial forms of the enzyme have been determined. Originally, the gene encoding galactokinase in humans was mapped to chromosome 17. An additional gene, encoding a protein with sequence similarity to galactokinase, was subsequently mapped to chromosome 15. Recent reports have shown that this second gene (GALK2) encodes an enzyme with greater activity against GalNAc than galactose. This enzyme, GalNAc kinase, has been implicated in a salvage pathway for the reutilization of free GalNAc derived from the degradation of complex carbohydrates. Here we report the first structural analysis of a GalNAc kinase. The structure of the human enzyme was solved in the presence of MnAMPPNP and GalNAc or MgATP and GalNAc (which resulted in bound products in the active site). The enzyme displays a distinctly bilobal appearance with its active site wedged between the two domains. The N-terminal region is dominated by a seven-stranded mixed beta-sheet, whereas the C-terminal motif contains two layers of anti-parallel beta-sheet. The overall topology displayed by GalNAc kinase places it into the GHMP superfamily of enzymes, which generally function as small molecule kinases. From this investigation, the geometry of the GalNAc kinase active site before and after catalysis has been revealed, and the determinants of substrate specificity have been defined on a molecular level. << Less

  J. Biol. Chem. 280:32784-32791(2005) [PubMed] [EuropePMC]

• Kidney N-acetylgalactosamine (GalNAc)-1-phosphate kinase, a new pathway of GalNAc activation.

  Pastuszak I., Drake R., Elbein A.D.

  A new enzyme that phosphorylates GalNAc at position 1 to form GalNAc-alpha-1P was purified approximately 1275-fold from the cytosolic fraction of pig kidney, and the properties of the enzyme were determined. The kinase is quite specific for GalNAc as the phosphate acceptor and is inactive with Glc ... >> More

  A new enzyme that phosphorylates GalNAc at position 1 to form GalNAc-alpha-1P was purified approximately 1275-fold from the cytosolic fraction of pig kidney, and the properties of the enzyme were determined. The kinase is quite specific for GalNAc as the phosphate acceptor and is inactive with GlcNAc, ManNAc, glucose, galactose, mannose, GalN, and GlcN. This enzyme is clearly separated from galactokinase by chromatography on phenyl-Sepharose. The GalNAc kinase has a pH optimum between 8.5 and 9.0 and requires a divalent cation in the order Mg2+ > Mn2+ > Co2+, with optimum Mg2+ concentration at approximately 5 mM. The enzyme was most active with ATP as the phosphate donor, but slight activity was observed with ITP, acetyl-P, and phosphoenolpyruvate. Enzyme activity was highest in porcine and human kidney and porcine liver, but was low in most other tissues. Cultured HT-29 cells also had high activity for this kinase. The purified enzyme fraction was incubated with azido-[32P]ATP, exposed to UV light, and run on SDS gels. A 50-kDa protein was labeled, and this labeling showed saturation kinetics with increasing amounts of the probe and was inhibited by unlabeled ATP. Although the most purified GalNAc kinase preparation still had two bands that labeled with ATP, maximum labeling of the 50-kDa protein, but not the 66-kDa band, was coincident with maximum GalNAc kinase activity on a column of DEAE-Cibacron blue. On Sephacryl S-300, the native enzyme has a molecular mass of 48-51 kDa, indicating that the active kinase is a monomer. The product of the reaction was characterized as GalNAc-alpha-1-P by various chemical procedures. << Less

  J Biol Chem 271:20776-20782(1996) [PubMed] [EuropePMC]

• Mechanistic studies on human N-acetylgalactosamine kinase.

  Agnew A., Timson D.

  N-Acetylgalactosamine kinase (GALK2) is a small molecule kinase from the GHMP family which phosphorylates N-acetylgalactosamine at the expense of ATP. Recombinant GALK2 expressed in, and purified from, Escherichia coli was shown to be active with the following kinetic parameters: Michaelis constan ... >> More

  N-Acetylgalactosamine kinase (GALK2) is a small molecule kinase from the GHMP family which phosphorylates N-acetylgalactosamine at the expense of ATP. Recombinant GALK2 expressed in, and purified from, Escherichia coli was shown to be active with the following kinetic parameters: Michaelis constant for ATP, 14 +/- 3 microM; Michaelis constant for N-acetylgalactosamine, 40 +/-14 microM; and turnover number, 1.0 +/-0.1 s(-1). The combination of substrate inhibition by N-acetylgalactosamine and alpha-methylgalactopyranoside acting as an uncompetitive inhibitor with respect to ATP suggested that the enzyme has an ordered ternary complex mechanism in which ATP is the first substrate to bind. The effects of pH on the kinetic parameters provided evidence for ionizable residues playing a role in substrate binding and catalysis. These results are discussed in the context of the mechanisms of the GHMP kinases. << Less

  J Enzyme Inhib Med Chem 25:370-376(2010) [PubMed] [EuropePMC]

• The intrinsic reactivity of ATP and the catalytic proficiencies of kinases acting on glucose, N-acetylgalactosamine, and homoserine: a thermodynamic analysis.

  Stockbridge R.B., Wolfenden R.

  To evaluate the rate enhancements produced by representative kinases and their thermodynamic basis, rate constants were determined as a function of changing temperature for 1) the spontaneous methanolysis of ATP and 2) reactions catalyzed by kinases to which different mechanisms of action have bee ... >> More

  To evaluate the rate enhancements produced by representative kinases and their thermodynamic basis, rate constants were determined as a function of changing temperature for 1) the spontaneous methanolysis of ATP and 2) reactions catalyzed by kinases to which different mechanisms of action have been ascribed. For each of these enzymes, the minor effects of changing viscosity indicate that k(cat)/K(m) is governed by the central chemical events in the enzyme-substrate complex rather than by enzyme-substrate encounter. Individual Arrhenius plots, obtained at intervals between pH 4.8 and 11.0, yielded Delta H(#) and T Delta S(#) for the nonenzymatic methanolysis of ATP(2-), ATP(3-), and ATP(4-) in the absence of Mg(2+). The addition of Mg(2+) led to partly compensating changes in Delta H(#) and T Delta S(#), accelerating the nonenzymatic methanolysis of ATP 11-fold at pH 7 and 25 degrees C. The rate enhancements produced by yeast hexokinase, homoserine kinase, and N-acetylgalactosamine kinase (obtained by comparison of their k(cat)/K(m) values in the presence of saturating phosphoryl acceptor with the second order rate constant for methanolysis of MgATP) ranged between 10(12)- and 10(14)-fold. Their nominal affinities for the altered substrates in the transition state were 2.1 x 10(-16) m for N-acetylgalactosamine kinase, 7.4 x 10(-17) m for homoserine kinase, and 6.4 x 10(-18) m for hexokinase. Compared with nonenzymatic phosphoryl transfer, all three kinases were found to produce major reductions in the entropy of activation, in accord with the likelihood that substrate juxtaposition and desolvation play prominent roles in their catalytic action. << Less

  J Biol Chem 284:22747-22757(2009) [PubMed] [EuropePMC]