Studies on hog spleen N-acetylglucosamine kinase. I. Purification and properties of N-acetylglucosamine kinase

Abstract

A highly specific kinase (ATP:2-acetylamino-2-deoxy-D-glucose 6-phospho-transferase) which phosphorylates 2-acetylamino-2-deoxy-D-glucose (GlcNAc) has been isolated from the extracts of hog spleen. The enzyme has been purified more than 3500-fold from the crude extract. Isolation and analysis procedures are described. The kinase reaction is optimal within a pH range of 8.6-9.4. The enzyme is strictly specific for GlcNAc as phosphate acceptor; ATP is the phosphoryl group donor for the kinase reaction and to a lesser extent GTP. K_m values for GlcNAc and ATP are 1.1 and 1.8 mM, respectively. The enzyme requires Mg²⁺, which may be replaced by other bivalent metal ions such as Mn² and Co²⁺ for lesser degrees of effectiveness. The enzyme is inhibited by P_i and PP_i. The enzyme is also inactivated by p-chloromercuribenzoate (PCMB) and the inactivation can be reversed by -SH compounds, e.g. cysteine and 2-mercaptoethanol. The enzyme is inhibited by the reaction products ADP and GlcNAc-6-P; ADP competes with ATP whereas the inhibition by GlcNAc-6-P is non-competitive. Other inhibitors studied are UTP, CTP and uridine diphosphate GlcNAc (UDP-GlcNAc), the end product of the pathway of GlcNAc metabolism.