The availability of both the structure of the galactokinase from L. lactis  and the readily expressible human form of the enzyme  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 kcat with no change in the specificity constant can only occur if Km drops by a similar factor to the turnover number. It is often (erroneously) assumed that Km 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 . 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 kcat, but a reduction in the Km for ATP means that the specificity constant for this substrate is not much changed compared to wild-type .
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 [21,22]). 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 ). 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 kcat. 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 . 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  and the first crystal structure of the enzyme  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 . 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 (Km,gal and kcat/Km,gal) suggesting that these residues formed part of, or were close to, the sugar binding site – a conclusion confirmed by the recent crystal structure .