Galactose is metabolised via the Leloir pathway 1 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 234. 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 5, yeast 67, plants 89 and mammals 101112. The primary sequence of these enzymes reveals only limited sequence similarity except at five highly conserved motifs 13. The first of these motifs – the so-called galactokinase signature motif – has been implicated in galactose binding 1213. Recent structural data 14 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) 23. 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 15. 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 16.

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 6. The rat liver enzyme can phosphorylate 2-deoxygalactose 11 and the enzyme from fenugreek seeds can use 2-deoxygalactose and fucose as substrates 8. 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 k_cat) with 2-deoxygalactose and 2-aminogalactose (but inactive with N-acetylgalactosamine) and weakly active (10 to 20-fold reduction in k_cat) with fucose 17. 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 71012. In contrast the plant enzyme has an ordered mechanism in which galactose binds first 8 and the enzyme from E. coli has a random mechanism in which either substrate can bind first 18.

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 19. 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.

The availability of both the structure of the galactokinase from L. lactis 14 and the readily expressible human form of the enzyme 12 mean that, for the first time, it is possible to interrogate the structure and function of the enzyme by site-directed mutagenesis. Here we report the results of some mutations to residues in motif I, which contributes most of the residues involved in galactose binding and recognition.

Elimination of the Glu-43 to C₆-OH hydrogen bond contact by mutating the side chain to alanine resulted in no major changes in the steady state kinetic parameters. In contrast altering this residue to glycine caused a substantial drop in the turnover number with no corresponding drop in the specificity constants. A reduction in k_cat with no change in the specificity constant can only occur if K_m drops by a similar factor to the turnover number. It is often (erroneously) assumed that K_m is a measure of the enzyme-substrate affinity. In fact it is a summed measure of the interaction between the substrate and the enzyme across all stages of the catalytic cycle (not just the initial enzyme-substrate encounter). In contrast, the specificity constant is a rate constant which does report on this initial encounter 20. Thus, in the case of E43G the kinetic constants tell us that the mutation causes little or no change in the rate of interaction between either ATP or galactose and the enzyme, that in at least one stage in the catalytic cycle the enzyme binds each substrate more tightly than the wild-type but that the overall rate of catalysis is reduced. A similar situation was observed in our studies of disease-causing mutants in human galactokinase – the mutant G346S which had a severely impaired k_cat, but a reduction in the K_m for ATP means that the specificity constant for this substrate is not much changed compared to wild-type 12.

It is interesting to note that although elimination of the Glu-43 side chain causes only modest changes to the kinetic parameters of galactokinase, whereas the elimination of the group it hydrogen bonds to in the sugar (C₆-OH) in either D-fucose or L-arabinose, results in the complete loss of detectable activity. Furthermore, these compounds are not inhibitors of the reaction, suggesting that they have essentially no affinity for the enzyme. This apparently paradoxical result can be explained by the different thermodynamic consequences of eliminating the charged and uncharged group in a charged hydrogen bond. Deletion of the uncharged group (i.e., in this case, the hydroxyl group on the sugar) leaves the charged carboxyl group in the protein unpaired. This is considerably energetically destabilised compared to the hydrogen bond (approximately 15 to 20 kJ.mol^-1 2122). In contrast, elimination of the charged group leaving the uncharged group is only slightly destabilised compared to the hydrogen bond (2 to 6 kJ.mol^-1 21). Mutating Glu-43 to glycine not only eliminates the charged component of the hydrogen bond, but also increases the flexibility of the peptide backbone. This increased mobility is the most likely explanation for the dramatic effects seen with this mutant on k_cat. A less rigid active site structure may well have a lower affinity for the transition state and, consequently, result in reduced activity.

Mutation of His-44 to either alanine or isoleucine resulted in insoluble protein following expression in E. coli. In our previous studies of the disease-causing mutations in human galactokinase, mutation of this residue to tyrosine resulted in an enzyme which was soluble but which was deficient in its interaction with galactose 12. Taken together this suggests that, in addition to any catalytic roles that this residue might play, it also has an important role in maintaining the structural integrity of the protein as a whole.

Although it might be expected that the D46A mutation would have similar consequences to E43A since both eliminated the charged group involved in two charged hydrogen bonds, it does not. Indeed this mutant has no detectable galactokinase activity. It is possible that this residue has important roles to play in forming the active site prior to galactose binding. Detailed speculation may therefore have to wait until the availability of a structure in the absence of a bound sugar molecule.

The combination of a readily expressible form of the human galactokinase 12 and the first crystal structure of the enzyme 14 mean that it is possible to design mutations in the enzyme to address specific questions. These results extend our studies on the enzyme which previously concentrated on point mutations which had been implicated in causing the genetic disease galactosemia 12. In that study we looked at several mutations either in motif I (G36R, H44Y), close to it in sequence (P28T, V32M) or close in space (G346S, G347S, G349S). Of these, three (P28T, V32M and G36R) proved to be insoluble on induction in E. coli suggesting that, like H44A, H44I and E43G/H44I in the current study these mutations affect the overall folding and stability of the protein. The soluble mutants (H44Y, G346S, G347S and G349S) all had effects on the constants which report on the interaction between the enzyme and galactose (K_m,gal and k_cat/K_m,gal) suggesting that these residues formed part of, or were close to, the sugar binding site – a conclusion confirmed by the recent crystal structure 14.

Recently, there has been a resurgence of interest in galactokinase as an enzyme and in the type galactosemia caused by its deficiency. If it does prove clinically viable to treat GALT-deficient galactosemia sufferers with galactokinase inhibiting drugs 16, the design of these drugs will require a thorough understanding of both the structure of the sugar binding site and how this site interacts with the substrate. Here we show that the enzyme will tolerate minor changes in the substrate at position 2 of the sugar ring, but not at positions 4 and 6. Alteration of residues by site directed mutagenesis which are close to, or in contact, with the sugar result in variety of effects ranging from insoluble protein through inactive enzyme to little observable change. These results could not have been predicted directly from the crystal structure and had to be determined experimentally. The combined approach of structural analysis and mutagenesis is likely to yield further insights in the coming months and years.

RJR conceived of and directed the project. DJT constructed (or supervised the construction of) the mutants, purified the proteins and carried out the kinetic assays and analysis. Both authors contributed to the interpretation of the data and have read and approved the contents of this manuscript.