Purification and properties of the adenosine diphosphate-glucose and uridine diphosphate-glucose pyrophosphorylases of Mycobacterium smegmatis: inhibition and activation of the adenosine diphosphate-glucose pyrophosphorylase.

Crude extracts of Mycobacterium smegmatis catalyzed the synthesis of adenosine diphosphate-glucose (ADP-Glc), cytidine diphosphate-glucose, guanosine diphosphate-glucose (GDP-Glc), thymidine diphosphate-glucose (TDP-Glc), and uridine diphosphate-glucose (UDP-Glc). In these crude enzyme fractions, high concentrations of trehalose-P inhibited the ADP-Glc and GDP-Glc pyrophosphorylases but did not effect the UDP-Glc or TDP-Glc pyrophosphorylases. Both the ADP-Glc pyrophosphorylase and the UDP-Glc pyrophosphorylase were partially purified (about 140-fold and 60-fold, respectively), and their properties were compared. For the ADP-Glc pyrophosphorylase, the K(m) for adenosine triphosphate was 6 x 10(-4)m, whereas that for glucose-1-P was 8 x 10(-4)m. The optimal concentration of Mg(2+) was 1 x 10(-3)m, and the pH optimum was 8.5. For the UDP-Glc pyrophosphorylase, the K(m) for uridine triphosphate was 1 x 10(-3)m and for glucose-1-P was 2 x 10(-3)m. The optimal Mg(2+) concentration was 1 x 10(-3)m, and the pH optimum was about 8.0. The purified ADP-Glc pyrophosphorylase was inhibited by fructose-6-P, fructose-1, 6-diphosphate, glucose-6-P, and phosphoenolpyruvate. On the other hand, trehalose, trehalose diphosphate, sodium pyruvate, and ribose-5-P did not effect the ADP-Glc pyrophosphorylase. None of these compounds, including trehalose-P, had any effect on the UDP-Glc pyrophosphorylase.