Human galactokinase shows high specificity at carbons 4 and 6 of the sugar
The ability of recombinant human galactokinase to catalyse the phosphorylation of sugars with structures similar to the natural substrate galactose was tested. No activity was observed with D-glucose, D-fucose, L-arabinose or N-acetyl-D-galactosamine, even when these sugars were present at high (100 mM) concentrations. Nor did any of these sugars act as inhibitors of the galactokinase reaction (data not shown). In contrast, 2-deoxy-D-galactose was a substrate for the enzyme (Fig. 3). The kinetic parameters were not much changed from those with galactose as substrate with no change greater than four-fold.
Mutation of glutamate 43 to alanine has little effect on the steady state kinetics
Since the elimination of the carbon-6 hydroxyl (in L-arabinose and D-fucose) results in a major reduction in the sugar-enzyme affinity we mutated glutamate 43 to alanine (the equivalent residue to Glu-42 in Fig. 1) in order to eliminate the side chain which contacts this part of the sugar. This mutant protein was soluble following expression in E. coli and could be purified. However, the yield obtained was approximately one third that of the wild type protein. Steady state kinetic analysis of the E43A mutant (Fig. 4) revealed few changes in the kinetic parameters (Table 1). This mutant, in common with the wild-type, has no detectable activity with D-fucose, L-arabinose and D-glucose as substrates.
Changing the sequence to match that of arabinose kinase results in insoluble protein
A double mutant E43G/H44I was constructed so as to alter the galactokinase sequence to that of arabinose kinase in the region which interacts with carbon 6. Although this protein could be expressed in E. coli, the majority was not in the soluble fraction following sonication of the bacterial cells. The small amount of protein that was soluble had minimal activity with D-galactose, D-glucose, L-arabinose or D-fucose (data not shown). Similar results were observed with the single mutants H44I and H44A. The single amino acid change, E43G does result in soluble, active protein (Fig. 4) although like E43A the yield is reduced on purification. This mutation causes an approximate ten-fold reduction in the turnover number (Table 1). However the specificity constants for both substrates are essentially unchanged compared to the wild-type.
Alteration of aspartate 46 results in an inactive enzyme
Mutation of the residue that contacts C3-OH and C4-OH on galactose results in a protein which can be readily expressed and purified from E. coli (the yield after purification was comparable to wild-type, data not shown). However, we were unable to detect any activity towards D-galactose, L-arabinose, D-fucose and D-glucose with this mutant, even at high concentrations of substrate (50 mM) and enzyme (0.48 μM enzyme compared to 0.067 μM with the E43A and E43G mutants).