title: Increased promiscuity of human galactokinase following alteration of a single amino acid residue distant from the active site.
abstract: Galactokinase catalyses the site- and stereospecific phosphorylation of galactose at the expense of ATP. The specificity of bacterial galactokinase enzymes can be broadened by alteration of a tyrosine residue to a histidine. The effects of altering the equivalent residue in human galactokinase (Tyr379) were investigated by testing all 19 possible variants. All of these alterations, except Y379P, resulted in soluble protein on expression in Escherichia coli and all the soluble variants could catalyse the phosphorylation of galactose, except Y379A and Y379E. The variants Y379C, Y379K, Y379R, Y379S and Y379W were all able to catalyse the phosphorylation of a variety of monosaccharides, including ones that are not acted on by the wild-type enzyme. Novel substrates for these variant galactokinases included D-mannose and D-fructose. The latter monosaccharide is presumed to react in the pyranose configuration. Molecular modelling suggested that the alterations do not cause changes to the overall structure of the enzyme. However, alteration of Tyr379 increases the flexibility of the peptide backbone in regions surrounding the active site. Therefore, it is proposed that alteration of Tyr379 affects the substrate specificity by the propagation of changes in flexibility to the active site, permitting a broader range of compounds to be accommodated.
authors: School of Biological Sciences, Queen's University, Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast, BT9 7BL, UK.
authors: Helena
authors: H
authors: Kristiansson
authors: David J
authors: DJ
authors: Timson
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D001665:Binding Sites; D005686:Galactokinase / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D005686:Galactokinase