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.

Improving the Activity and Stability of Human Galactokinase for Therapeutic and Biotechnological Applications

“…MD simulations of these variants revealed some interesting features when compared to the wild‐type proteins. In 2 , alterations were seen in a largely α‐helical region (residues 232–240) that has previously been implicated in catalysis . Another key change was observed around residue 156 and resulted in the shortening of an α‐helix and consequent displacement of residues 163–177 (Figure A).…”

“…The MD studies also re‐emphasise the critical role of residues 232–240 in the structure, dynamics and catalytic activity of the enzyme. We previously identified this as a critical region for the catalytic mechanism of the enzyme . We hypothesise that direct alteration of these residues might affect the activity or specificity of the kinase.…”

“…Expression, purification and mutagenesis of human GALK1 : Recombinant human wild‐type galactokinase was expressed in, and purified from, E. coli as previously described . For site‐directed mutagenesis, the plasmid was mutated by using the QuikChange method, and the full sequence was verified (GATC Biotech, London, UK) .…”

“…Kinetic constants were determined by arraying reactions in an eight by ten grid in order to determine apparent Michaelis constants ( K m,app ) and apparent turnover numbers ( k cat,app ) over a range of subsaturating concentrations of ATP and galactose by using nonlinear curve fitting to the Michaelis–Menten equation, as implemented in GraphPad Prism 6.0 (GraphPad Software, CA, USA). These were then used to determine the absolute values of the turnover number ( k cat ), the Michaelis constant for galactose ( K m,gal ) and the Michaelis constant for ATP ( K m,ATP ), as previously described . In brief, plots of k cat.app against substrate concentration were fitted by using Equations a) and b) to determine absolute values. …”

“…Analytical methods : Protein concentrations were estimated by the method of Bradford with bovine serum albumin as a standard . The thermal stability of galactokinase was measured by differential scanning fluorimetry (DSF) with Sypro Orange as the fluorescent probe, as previously described . Native gel electrophoresis was used to determine the proteins’ resistance to chaotropic denaturation, as previously described …”