Type III
galactosemia results from reduced activity of the
enzyme UDP-galactose 4'-epimerase. Five disease-associated alleles (G90E, V94M, D103G, N34S and L183P) and three artificial alleles (Y105C, N268D, and M284K) were tested for their ability to alleviate
galactose-induced growth arrest in a Saccharomyces cerevisiae strain which lacks endogenous
UDP-galactose 4'-epimerase. For all of these alleles, except M284K, the ability to alleviate
galactose sensitivity was correlated with the
UDP-galactose 4'-epimerase activity detected in
cell extracts. The M284K allele, however, was able to substantially alleviate
galactose sensitivity, but demonstrated near-zero activity in
cell extracts. Recombinant expression of the corresponding
protein in Escherichia coli resulted in a
protein with reduced enzymatic activity and reduced stability towards denaturants in vitro. This lack of stability may result from the introduction of an unpaired positive charge into a bundle of three α-helices near the surface of the
protein. The disparities between the in vivo and in vitro data for M284K-hGALE further suggest that there are additional, stabilising factors present in the cell. Taken together, these results reinforce the need for care in the interpretation of in vitro, enzymatic diagnostic tests for type III
galactosemia.