title: Insight into the mechanism of galactokinase: Role of a critical glutamate residue and helix/coil transitions.
abstract: 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.
authors: School of Biological Sciences, Queen's University Belfast, Medical Biology Building, 97 Lisburn Road, Belfast BT9 7BL, UK.
authors: Margaret
authors: M
authors: McAuley
authors: School of Chemistry and Chemical Engineering, Queen's University Belfast, David Keir Building, Stranmillis Road, Belfast BT9 5AG, UK.
authors: Meilan
authors: M
authors: Huang
authors: School of Biological Sciences, Queen's University Belfast, Medical Biology Building, 97 Lisburn Road, Belfast BT9 7BL, UK; School of Pharmacy and Biomolecular Sciences, University of Brighton, Huxley Building, Lewes Road, Brighton BN2 4GJ, UK. Electronic address: d.timson@brighton.ac.uk.
authors: David J
authors: DJ
authors: Timson
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001224:Aspartic Acid / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain / Q000502:physiology; D005686:Galactokinase / Q000378:metabolism*; D005690:Galactose / Q000378:metabolism; D005693:Galactosemias / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism*; D006801:Humans; D008715:Methionine / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D001224:Aspartic Acid; D018698:Glutamic Acid; D000255:Adenosine Triphosphate; D008715:Methionine; D005686:Galactokinase; D005690:Galactose
keywords: Active site; Biocatalysis; GALK1; Galactosemia; Molecular dynamics; Protein flexibility