Galactose is metabolised via the Leloir pathway  in order to produce glucose-6-phosphate that can enter glycolysis. The first committed step of this pathway is the phosphorylation of galactose at the expense of ATP – a reaction that is catalysed by the enzyme galactokinase. Lack of functional galactokinase in humans is one cause of the inherited disease galactosemia [2-4]. The main symptom of this disease, which is treatable by the complete removal of lactose and galactose from the diet, is early onset cataracts. The enzyme has been purified and characterised from a variety of different sources including bacteria , yeast [6,7], plants [8,9] and mammals [10-12]. The primary sequence of these enzymes reveals only limited sequence similarity except at five highly conserved motifs . The first of these motifs – the so-called galactokinase signature motif – has been implicated in galactose binding [12,13]. Recent structural data  on the galactokinase from the bacterium Lactococcus lactis confirms this hypothesis and shows that most of the contacts between the sugar and the protein are provided by residues in this motif (Fig. 1). The cavity in which galactose binds is, in part, defined by a histidine residue (H43 in L. lactis which is equivalent to H44 in the human enzyme). The side-chain of this residue is located close to, but not in contact with, the hydroxyl attached to carbon 6 of the sugar.
Another, and more common, cause of galactosemia is deficiency of the next enzyme in the Leloir pathway, galactose-1-phosphate uridyl transferase (GALT) [2,3]. The symptoms of this deficiency are generally more severe and include, in addition to cataracts, damage to the brain, liver and kidneys – effects which cannot be reversed or even completely prevented by the exclusion of galactose and lactose from the diet. This increased severity is believed to result from the build up of the toxic metabolite galactose-1-phosphate. The mechanism of galactose-1-phosphate toxicity is not known. However, in brain at least it may be linked to the substantial (five-fold in conditions designed to mimic those observed in GALT-deficient patients) increases in Mg-ATPase activity and consequent depletion of ATP within the cell . Recently, it has been suggested that inhibition of galactokinase in GALT-deficient patients might be used in addition to diet to prevent the build up of galactose-1-phosphate and thus the development of the more severe symptoms .
The yeast enzyme is reported as having high specificity for the sugar substrate with no ability to phosphorylate glucose, mannose, galactitol, arabinose, 2-deoxygalactose, fucose or lactose . The rat liver enzyme can phosphorylate 2-deoxygalactose  and the enzyme from fenugreek seeds can use 2-deoxygalactose and fucose as substrates . A detailed study of the substrate specificity of the E. coli galactokinase showed that the enzyme was moderately active (less than 10-fold reduction in kcat) with 2-deoxygalactose and 2-aminogalactose (but inactive with N-acetylgalactosamine) and weakly active (10 to 20-fold reduction in kcat) with fucose . The enzymes from rat liver and yeast have the same kinetic mechanism as the human one – an ordered ternary complex mechanism in which ATP is the first substrate to bind [7,10,12]. In contrast the plant enzyme has an ordered mechanism in which galactose binds first  and the enzyme from E. coli has a random mechanism in which either substrate can bind first .
No sugar specificity study has yet been carried out with the human enzyme. The variety of substrate specificities and the diversity of reaction mechanisms mean that it is imperative that studies on the human enzyme be carried out in order to inform, accurately, any future study of therapeutically useful inhibitors of the galactokinase reaction. We were particularly interested in sugars that differ at carbons 4 and 6 – parts of the molecule which make hydrogen bonds with the protein. D-Glucose differs from D-galactose only in the configuration of the hydroxyl group at position 4 (Fig 2). D-fucose (6-deoxy-D-galactose) differs from galactose in that it lacks a hydroxyl group at position 6. L-arabinose lacks carbon 6 (and its associated hydroxyl) altogether.
The importance of the aspartate and histidine residues in the recognition of the sugar is underlined by the observation that the related sugar kinase, arabinose kinase, has an almost identical sequence in motif I except that these two residues are altered to glycine and isoleucine, respectively . We complemented our sugar specificity studies by mutating three key, conserved residues (Glu-43, His-44 and Asp-46) in the sugar binding site of the human galactokinase and assessing the kinetic consequences of these changes. Interestingly although the abolition of the carbon 6 hydroxyl in arabinose and fucose results in no activity, deleting the side chain which makes a hydrogen bond with this part of the sugar causes little change in the steady state kinetics of the galactokinase reaction.