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 , 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.