Laura Shen

Biosynthesis of Bacterial Glycogen

Abstract Adenosine diphosphate-glucose pyrophosphorylase from Arthrobacter viscosus NRRL B1973 was found to be activated by fructose-6-P, pyruvate, deoxyribose-5-P and ribose-5-P. Fructose-6-P was the most effective activator. Fructose-6-P increased the Vmax of pyrophosphorolysis of ADP-glucose 2.5-fold, and the Vmax of synthesis of ADP-glucose from ATP and glucose-1-P, 7-fold. The apparent Kms for ATP, pyrophosphate, and ADP-glucose were decreased in the presence of fructose-6-P. Pyruvate increased the Vmax of pyrophosphorolysis 2-fold and of synthesis 6-fold. However, pyruvate only decreased the Km for pyrophosphate. The Km for glucose-1-P was not effected by either pyruvate or fructose-6-P. The enzyme was found to be inhibited by phosphate, sulfate, AMP, ADP, phosphoenolpyruvate, GMP, and GDP. However, fructose-6-P completely overcame (reversed) these inhibitions. The relationship of these findings to the control of bacterial glycogen synthesis is discussed.

[42] ADP-glucose pyrophosphorylase from Arthrobacter

Publisher Summary This chapter discusses the synthesis of adenosine diphosphate (ADP)-glucose pyrophosphorylase from Arthrobacter . Enzymatic activity is determined by measuring the synthesis of adenosine triphosphate (ATP)- 32 P from ADP-glucose and P- 32 P i . The ATP is isolated by adsorption onto Norit A and estimated by measuring the radioactivity contained in the Norit. The reagents used, procedure followed, and steps involved in the purification are also described in the chapter. The assay is employed in a number of bacterial extracts, and reasonably good proportionality between enzyme activity and amount of protein is obtained. Potassium fluoride (KF) is added to the reaction mixture to inhibit the inorganic pyrophosphatase in the crude extracts and is not required in the purified enzyme. The pyrophosphorylase contains no detectable amounts of phosphoglucomutase, adenosine triphosphatse (ATPase), aldolase, inorganic pyrophosphatase, and phosphohexoisomerase. Magnesium ++ (Mg ++ ) is necessary for enzyme action. Maximal activity is obtained at 6 × 10 –3 M. The enzyme converts glucose- 14 C-1-P quantitatively into ADP-glucose- 14 C if activator and inorganic pyrophosphatase are added to the reaction mixture.