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.
D000241:Adenosine; D000645:Ammonium Sulfate; D002130:Calcium Phosphates; D002474:Cell-Free System; D011232:Chemical Precipitation; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004789:Enzyme Activation / Q000187:drug effects; D005636:Fructosephosphates / Q000494:pharmacology; D005964:Glucosyltransferases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D006600:Hexosephosphates / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006882:Hydroxyapatites; D008274:Magnesium / Q000494:pharmacology; D009161:Mycobacterium / Q000201:enzymology*; D009702:Nucleoside Diphosphate Sugars; D010728:Phosphoenolpyruvate / Q000494:pharmacology; D014539:Uridine Diphosphate Sugars
D002130:Calcium Phosphates; D005636:Fructosephosphates; D006600:Hexosephosphates; D006882:Hydroxyapatites; D009702:Nucleoside Diphosphate Sugars; D014539:Uridine Diphosphate Sugars; D010728:Phosphoenolpyruvate; D005964:Glucosyltransferases; D008274:Magnesium; D000241:Adenosine; D000645:Ammonium Sulfate