The role of the active site residues in human galactokinase: Implications for the mechanisms of GHMP kinases

Clare F. Megarity, Meilan Huang, Claire Warnock, David J. Timson

Research output: Contribution to journalArticlepeer-review

Abstract

Galactokinase catalyses the phosphorylation of galactose at the expense of ATP. Like other members of the GHMP family of kinases it is postulated to function through an active site base mechanism in which Asp-186 abstracts a proton from galactose. This asparate residue was altered to alanine and to asparagine by site-directed mutagenesis of the corresponding gene. This resulted in variant enzyme with no detectable galactokinase activity. Alteration of Arg-37, which lies adjacent to Asp-186 and is postulated to assist the catalytic base, to lysine resulted in an active enzyme. However, alteration of this residue to glutamate abolished activity. All the variant enzymes, except the arginine to lysine substitution, were structurally unstable (as judged by native gel electrophoresis in the presence of urea) compared to the wild type. This suggests that the lack of activity results from this structural instability, in addition to any direct effects on the catalytic mechanism. Computational estimations of the pKa values of the arginine and aspartate residues, suggest that Arg-37 remains protonated throughout the catalytic cycle whereas Asp-186 has an abnormally high pKa value (7.18). Quantum mechanics/molecular mechanics (QM/MM) calculations suggest that Asp-186 moves closer to the galactose molecule during catalysis. The experimental and theoretical studies presented here argue for a mechanism in which the C 1-OH bond in the sugar is weakened by the presence of Asp-186 thus facilitating nucleophilic attack by the oxygen atom on the γ-phosphorus of ATP.

Original languageEnglish
Pages (from-to)120-126
Number of pages7
JournalBioorganic Chemistry
Volume39
Issue number3
DOIs
Publication statusPublished - 1 Jun 2011

Keywords

  • Active site base
  • Enzyme mechanism
  • Galactokinase
  • Galactosemia
  • GHMP kinase
  • Protein stability

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