Dual Role of Hexose-1-phosphate Uridylyltransferase in Galactosamine Metabolism

The metabolism of 2-amino-2-deoxy-d-galactose (galactosamine) in liver involves the formation of UDP-galactosamine, UDP-glucosamine, UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine; however, the conversion of the UDP-hexosamines to the UDP-N-acetylhexosamines requires explanation. We have therefore performed kinetic studies on hexose-1-phosphate uridylyltransferase from liver at pH 7.4 and 37°C in order to analyse whether this enzyme catalyzes the synthesis of glucosamine 1-phosphate as an obligatory intermediate in the formation of UDP-N-acetylglucosamine. Glucosamine 1-phosphate production was demonstrated using enzymatically synthesized UDP-glucosamine that substituted for UDP-glucose in the presence of galactose 1-phosphate or galactosamine 1-phosphate. The separation of UDP-glucosamine and UDP-galactosamine, a prerequisite in these studies, was achieved by anion-exchange high-performance liquid chromatography based on the preferential borate complex formation of UDP-galactosamine. At fixed UDP-glucosamine concentrations (0.4 and 0.5 mmol/l) the apparent Km values for galactosamine 1-phosphate and galactose 1-phosphate were 0.53 and 0.02 mmol/l, respectively. Both Km values increased about 20-fold when UDP-glucose served as the constant cosubstrate. Apparent Km values for UDP-glucosamine at fixed levels of galactosamine 1-phosphate (2 mmol/l) and galactose 1-phosphate (0.08 mmol/l) were 0.25 and 0.31 mmol/l, respectively. These Km values are in the range of concentrations determined in vivo earlier in rat liver after a hepatotoxic dose of galactosamine. The estimated velocity of hexose-1-phosphate uridylyltransferase in liver in the presence of the galactosamine-derived substrates can account for the observed increment of UDP-N-acetylhexosamines. Uridylyltransferase fulfills a dual role in galactosamine metabolism by the activation of galactosamine 1-phosphate to UDP-galactosamine and, after epimerization to UDP-glucosamine, by the formation of glucosamine 1-phosphate as the precursor of UDP-N-acetylhexosamines.