Inhibition of 6-phosphofructo-2-kinase activity by mercaptopurines.

The activity of 6-phosphofructo-2-kinase (PFK-2), the enzyme that catalyses the synthesis of fructose 2,6-bisphosphate (Fru-2,6-P2), was inhibited by mercaptopurines in vitro. Inhibition was observed with the purified enzyme from rat liver and bovine heart, and in extracts from rat lymphocytes and hepatoma cells, chick embryo fibroblasts, and human HeLa and lymphoblastoid cells. Half-maximal effect was obtained with 0.1-0.2 mM mercaptopurine and maximal inhibition ranged between 50 and 90% depending on the enzyme preparation. The inhibition resulted from a decrease in Vmax with no change in Km for ATP. The inhibition was relieved by treatment of the enzyme with thiol reducing agents, suggesting that it involves the formation of a mixed disulfide between mercaptopurine and thiol group(s) essential for enzyme activity. Incubation of intact lymphocytes or lymphoblastoid cells with 2- or 6-mercaptopurine resulted in a decrease in Fru-2,6-P2 content and lactate release. A decrease in Fru-2,6-P2 content but no change in lactate release was observed in HeLa cells and fibroblasts treated with 6-mercaptopurine but not with 2-mercaptopurine. Treatment of HeLa cells with 6-mercaptopurine resulted in a decreased PFK-2 activity which could be restored by treatment of the cell extract with dithiothreitol. In isolated rat hepatocytes and perfused rat hearts mercaptopurines had little or no effect on the Fru-2,6-P2 content and lactate release. These results suggest that the effect of 6-mercaptopurine of arresting growth in lymphoid cells might involve the inhibition of glycolysis in addition to the known inhibition of de novo purine nucleotide synthesis.