Functional analysis of disease-causing mutations in human UDP-galactose 4-epimerase

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Abstract

UDP-galactose 4-epimerase (GALE, EC 5.1.3.2) catalyses the interconversion of UDP-glucose and UDP-galactose. Point mutations in this enzyme are associated with the genetic disease, type III galactosemia, which exists in two forms - a milder, or peripheral, form and a more severe, or generalized, form. Recombinant wild-type GALE, and nine disease-causing mutations, have all been expressed in, and purified from, Escherichia coli in soluble, active forms. Two of the mutations (N34S and G319E) display essentially wild-type kinetics. The remainder (G90E, V94M, D103G, L183P, K257R, L313M and R335H) are all impaired in turnover number (kcat) and specificity constant (kcat/K m), with G90E and V94M (which is associated with the generalized form of galactosemia) being the most affected. None of the mutations results in a greater than threefold change in the Michaelis constant (Km). Protein-protein crosslinking suggests that none of the mutants are impaired in homodimer formation. The L183P mutation suffers from severe proteolytic degradation during expression and purification. N34S, G90E and D103G all show increased susceptibility to digestion in limited proteolysis experiments. Therefore, it is suggested that reduced catalytic efficiency and increased proteolytic susceptibility of GALE are causative factors in type III galactosemia. Furthermore, there is an approximate correlation between the severity of these defects in the protein structure and function, and the symptoms observed in patients.

Original languageEnglish
Pages (from-to)6170-6177
Number of pages8
JournalFEBS Journal
Volume272
Issue number23
DOIs
Publication statusPublished - 1 Dec 2005

Keywords

  • Galactosemia
  • GALE
  • Leloir pathway
  • SDR family enzyme
  • UDP-glucose

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