Molecular basis for severe epimerase deficiency galactosemia - X-ray structure of the human V94M-substituted UDP-galactose 4-epimerase

Galactosemia is an inherited disorder characterized by an inability to meta bolize galactose. Although classical galactosemia results from impairment o f the second enzyme of the Leloir pathway, namely galactose-1-phosphate uri dylyltransferase, alternate forms of the disorder can occur due to either g alactokinase or UDP-galactose 4-epimerase deficiencies. One of the more sev ere cases of epimerase deficiency galactosemia arises from an amino acid su bstitution at position 94. It has been previously demonstrated that the V94 M protein is impaired relative to the wild-type enzyme predominantly at the level of V-max rather than K-m. To address the molecular consequences the mutation imparts on the three-dimensional architecture of the enzyme, we ha ve solved the structures of the V94M-substituted human epimerase complexed with NADH and UDP-glucose, UDP-galactose, UDP-GlcNAc, or UDP-GalNAc. In the wild-type enzyme, the hydrophobic side chain of Val(94) packs near the aro matic group of the catalytic Tyr(157) and serves as a molecular "fence" to limit the rotation of the glycosyl portions of the UDP-sugar substrates wit hin the active site. The net effect of the V94M substitution is an opening up of the Ala(93) to Glu(96) surface loop, which allows free rotation of th e sugars into nonproductive binding modes.