TY - JOUR
T1 - Insight into the mechanism of galactokinase: role of a critical glutamate residue and helix/coil transitions
AU - McAuley, Margaret
AU - Huang, Meilan
AU - Timson, David J.
PY - 2016/10/24
Y1 - 2016/10/24
N2 - Galactokinase, the enzyme which catalyses the first committed step in the Leloir pathway, has
attracted interest due to its potential as a biocatalyst and as a possible drug target in the treatment
of type I galactosemia. The mechanism of the enzyme is not fully elucidated. Molecular dynamics
(MD) simulations of galactokinase with the active site residues Arg-37 and Asp-186 altered predicted
that two regions (residues 174-179 and 231-240) had different dynamics as a consequence.
Interestingly, the same two regions were also affected by alterations in Arg-105, Glu-174 and Arg-
228. These three residues were identified as important in catalysis in previous computational studies
on human galactokinase. Alteration of Arg-105 to methionine resulted in a modest reduction in
activity with little change in stability. When Arg-228 was changed to methionine, the enzyme’s
interaction with both ATP and galactose was affected. This variant was significantly less stable than
the wild-type protein. Changing Glu-174 to glutamine (but not to aspartate) resulted in no
detectable activity and a less stable enzyme. Overall, these combined in silico and in vitro studies
demonstrate the importance of a negative charge at position 174 and highlight the critical role of the
dynamics in to key regions of the protein. We postulate that these regions may be critical for
mediating the enzyme’s structure and function.
AB - Galactokinase, the enzyme which catalyses the first committed step in the Leloir pathway, has
attracted interest due to its potential as a biocatalyst and as a possible drug target in the treatment
of type I galactosemia. The mechanism of the enzyme is not fully elucidated. Molecular dynamics
(MD) simulations of galactokinase with the active site residues Arg-37 and Asp-186 altered predicted
that two regions (residues 174-179 and 231-240) had different dynamics as a consequence.
Interestingly, the same two regions were also affected by alterations in Arg-105, Glu-174 and Arg-
228. These three residues were identified as important in catalysis in previous computational studies
on human galactokinase. Alteration of Arg-105 to methionine resulted in a modest reduction in
activity with little change in stability. When Arg-228 was changed to methionine, the enzyme’s
interaction with both ATP and galactose was affected. This variant was significantly less stable than
the wild-type protein. Changing Glu-174 to glutamine (but not to aspartate) resulted in no
detectable activity and a less stable enzyme. Overall, these combined in silico and in vitro studies
demonstrate the importance of a negative charge at position 174 and highlight the critical role of the
dynamics in to key regions of the protein. We postulate that these regions may be critical for
mediating the enzyme’s structure and function.
U2 - 10.1016/j.bbapap.2016.10.012
DO - 10.1016/j.bbapap.2016.10.012
M3 - Article
SN - 1570-9639
JO - Biochimica et Biophysica Acta - Proteins and Proeomics
JF - Biochimica et Biophysica Acta - Proteins and Proeomics
ER -