Mechanistic studies on human N-acetylgalactosamine kinase

Andrew Agnew, David Timson

Research output: Contribution to journalArticlepeer-review

Abstract

N-Acetylgalactosamine kinase (GALK2) is a small molecule kinase from the GHMP family which phosphorylates N-acetylgalactosamine at the expense of ATP. Recombinant GALK2 expressed in, and purified from, Escherichia coli was shown to be active with the following kinetic parameters: Michaelis constant for ATP, 14±3μM; Michaelis constant for N-acetylgalactosamine, 40±14μM; and turnover number, 1.0±0.1s-1. The combination of substrate inhibition by N-acetylgalactosamine and α-methylgalactopyranoside acting as an uncompetitive inhibitor with respect to ATP suggested that the enzyme has an ordered ternary complex mechanism in which ATP is the first substrate to bind. The effects of pH on the kinetic parameters provided evidence for ionizable residues playing a role in substrate binding and catalysis. These results are discussed in the context of the mechanisms of the GHMP kinases.

Original languageEnglish
Pages (from-to)370-376
Number of pages7
JournalJournal of Enzyme Inhibition and Medicinal Chemistry
Volume25
Issue number3
DOIs
Publication statusPublished - 30 Apr 2010

Keywords

  • Enzyme mechanism
  • Galactokinase
  • GALK2
  • GHMP kinase
  • N-acetylgalactosamine
  • PH study

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