Mechanistic studies on human N-acetylgalactosamine kinase

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 ± 3 μM; Michaelis constant for N-acetylgalactosamine, 40 ± 14 μM; and turnover number, 1.0 ± 0.1 s⁻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.

Keywords::

• GHMP kinase
• N-acetylgalactosamine
• pH study
• enzyme mechanism
• galactokinase
• GALK2

Acknowledgements

We thank Peter McColgin and Andrew Frazer who carried out preliminary work on human GALK2 and Prof Hazel Holden (University of Wisconsin) who provided the GALK2 expression construct.

Declaration of interest: This work was funded in part by a grant from the Royal Society, UK (2004/R1).