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 microM; Michaelis constant for N-acetylgalactosamine, 40 +/- 14 microM; and turnover number, 1.0 +/- 0.1 s(-1). The combination of substrate inhibition by N-acetylgalactosamine and alpha-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.