Studies on hog spleen N-acetylglucosamine kinase : II. Allosteric regulation of the activity of N-acetylglucosamine kinase

Abstract

Kinetic studies of hog spleen N-acetylglucosamine kinase indicate that N-acetylglucosamine-6-phosphate (GlcNAc-6-p), the product of the reaction and UDP-N-acetylglycosamine (UDP-GlcNAc), the end product of the pathway of N-acetylglucosamine (GlcNAc) metabolism, are noncompetitive inhibitors. Maximum inhibitory effect of these metabolites occurs around pH 7.5, whereas the kinase reaction is optimal within a pH range of 8.6-9.4. Binding of these inhibitors with the enzyme shows cooperative homotropic interactions. Hill plot of GlcNAc saturation kinetics of the enzyme which yielded an interaction coefficient of n = 1.66 suggests the presence of at least two binding sites for the substrate on the enzyme molecule. The allosteric effect of GlcNAc-6-p and UDP-GlcNAc was confirmed since specific disruption of the inhibitor-binding site(s) is possible without much loss of the catalytic activity of the enzyme. The desensitization of N-acetyl glucosamine kinase to its inhibitors was brought about by controlled heat treatment. The selective destruction of the effect of those inhibitors on the kinase strongly suggests that the site for binding the inhibitors is largely independent of the site for binding the substrates. All of these results strongly suggest that the hog spleen N-acetylglucosamine kinase is a regulatory enzyme, and the end product UDP-GlcNAc and the reaction product GlcNac-6-p both act on the enzyme as feedback inhibitor.