Molecular structure of human galactokinase: implications for type II galactosemia

Galactokinase functions in the Leloir pathway for galactose metabolism by catalyzing the MgATP-dependent phosphorylation of the C-1 hydroxyl group of alpha-D-galactose. The enzyme is known to belong to the GHMP superfamily of small molecule kinases and has attracted significant research attention fo...

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Bibliographic Details
Published in:The Journal of biological chemistry 2005-03, Vol.280 (10), p.9662
Main Authors: Thoden, James B, Timson, David J, Reece, Richard J, Holden, Hazel M
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Binding Sites
Cloning, Molecular
Disulfides - analysis
Escherichia coli - genetics
Galactokinase - chemistry
Galactokinase - genetics
Galactose - metabolism
Galactosemias - genetics
Humans
Mutagenesis, Site-Directed
Protein Conformation
Protein Structure, Secondary
Recombinant Proteins - chemistry
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Summary:Galactokinase functions in the Leloir pathway for galactose metabolism by catalyzing the MgATP-dependent phosphorylation of the C-1 hydroxyl group of alpha-D-galactose. The enzyme is known to belong to the GHMP superfamily of small molecule kinases and has attracted significant research attention for well over 40 years. Approximately 20 mutations have now been identified in human galactokinase, which result in the diseased state referred to as Type II galactosemia. Here we report the three-dimensional architecture of human galactokinase with bound alpha-D-galactose and Mg-AMPPNP. The overall fold of the molecule can be described in terms of two domains with the active site wedged between them. The N-terminal domain is dominated by a six-stranded mixed beta-sheet whereas the C-terminal motif contains six alpha-helices and two layers of anti-parallel beta-sheet. Those residues specifically involved in sugar binding include Arg37, Glu43, His44, Asp46, Gly183, Asp186, and Tyr236. The C-1 hydroxyl group of alpha-D-galactose sits within 3.3 A of the gamma-phosphorus of the nucleotide and 3.4 A of the guanidinium group of Arg37. The carboxylate side chain of Asp186 lies within approximately 3.2 A of the C-2 hydroxyl group of alpha-D-galactose and the guanidinium group of Arg37. Both Arg37 and Asp186 are strictly conserved among both prokaryotic and eukaryotic galactokinases. In addition to providing molecular insight into the active site geometry of the enzyme, the model also provides a structural framework upon which to more fully understand the consequences of the those mutations known to give rise to Type II galactosemia.
ISSN:0021-9258
DOI:10.1074/jbc.M412916200