Time-dependent inhibition of glucose 6-phosphatase by 3-mercaptopicolinic acid

3-Mercaptopicolinate (3-MP) inhibits D-glucose-6-phosphate (G6P) phosphohydrolase activity of the glucose-6-phosphatase system (Bode et al. (1993) Biochem. Cell Biol. 71, 113-121). We therefore attempted to maximize the inhibition by varying the physical state of microsomes, the concentration of 3-MP, and the time of preliminary incubation of 3-MP with the enzyme. The inhibition was irreversible and time- and inhibitor-concentration-dependent, with G6P phosphohydrolase activity of intact rat liver microsomes, but there was no inhibition with detergent-treated microsomes. The effectiveness of 3-MP as a time-dependent inhibitor of glucose 6-phosphatase was demonstrated in situ by measuring glycogenolysis in isolated, perfused livers from fed rats. We first exposed the livers to 2 mM 3-MP for 40 min, and then assessed the inhibitory effects on glycogenolysis. It was lowered by 50%. These observations establish that 3-MP at the mM level may be useful as an experimental probe in the study of the role(s) of G6P in the regulation of glycogenolysis as well as glycogenesis. Further, they validate the use of much lower (microM) concentrations of 3-MP to block gluconeogenesis (at the phosphoenolpyruvate carboxykinase step) without interfering with glucose 6-phosphatase. We also explored the mechanism of 3-MP inhibition. The time-dependent inhibition of carbamoyl-phosphate:glucose phosphotransferase activity with microsomes incubated with 1 mM 3-MP for 60 or 90 min and then assayed with 1 mM carbamoyl phosphate and 180 mM glucose was modest compared with inhibition of G6P phosphohydrolase. When G6P production by carbamoyl-phosphate:glucose phosphotransferase was reduced by decreasing glucose concentration to 60 mM, no inhibition by 3-MP was discernible. There was no inhibition of inorganic pyrophosphatase activity. These studies support the model of time-dependent, irreversible reaction of 3-MP with the G6P translocase component of the glucose-6-phosphatase system.