Studies of the V94M-substituted human UDPgalactose-4-epimerase enzyme associated with generalized epimerase-deficiency galactosaemia

Impairment of the human enzyme UDPgalactose 4-epimerase (hGALE) results in epimerase-deficiency galactosaemia, an inborn error of metabolism with vari able biochemical presentation and clinical outcomes reported to range from benign to severe. Molecular studies of the hGALE loci from patients with ep imerase deficiency reveal significant allelic heterogeneity, raising the po ssibility that variable genotypes may constitute at least one factor contri buting to the biochemical and clinical heterogeneity observed. Previously w e have identified a single substitution mutation, V94M, present in the homo zygous state in all patients genotyped with the severe, generalized form of epimerase-deficiency galactosaemia. We report here further studies of the V94M-hGALE enzyme, overexpressed and purified from a null-background yeast expression system. Our results demonstrate that the mutant protein is impai red relative to the wild-type enzyme predominantly at the level of V-max ra ther than of K-m. Studies using UDP-N-acetylgalactosamine as a competitor o f UDPgalactose further demonstrate that the K-m values for these two substr ates vary by less than a factor of 3 for both the wild-type and mutant prot eins. Finally, we have explored the impact of the V94M substitution on susc eptibility of yeast expressing human GALE to galactose toxicity, including changes in the levels of galactose 1-phosphate (gal-1-P) accumulated in the se cells at different times following exposure to galactose. We have observ ed an inverse correlation between the level of GALE activity expressed in a given culture and the degree of galactose toxicity observed. We have furth er observed an inverse correlation between the level of GALE activity expre ssed in a culture and the concentration of gal-1-P accumulated in the cells . These data support the hypothesis that elevated levels of gal-1-P may und erlie the observed toxicity. They further raise the intriguing possibility that yeast may provide a valuable model not only for assessing the impact o f given patient mutations on hGALE function, but also for exploring the met abolic imbalance resulting from impaired activity of GALE in living cells.