The biosynthesis of granulose by Clostridium pasteurianum.
1. Mutant strains of Clostridium pasteurianum were obtained, which are unable to synthesize granulose (an intracellularly accumulated amylopectin-like alpha-polyglucan). 2. These mutants lacked either (a) ADP-glucose pyrophosphorylase (EC 2.7.7.27), or (b) granulose synthase (i.e. ADP-glucose-alpha-1,4-glucan glucosyltransferase, EC 2.4.1.21). 3. Although both of these enzymes were constitutively synthesized by the wild-type organism, massive deposition of granulose in a sporulating culture coincided with a threefold increase in the specific activity of ADP-glucose pyrophosphorylase. 4. The soluble ADP-glucose pyrophosphorylase was partially purified (33-fold). Its ATP-saturation curve was not sigmoidal and its activity was not enhanced by phosphorylated intermediates of glycolysis, pyruvate, NAD(P)H or pyridoxal 5'-phosphate. ADP at relatively high concentrations acted as a competitive inhibitor (K(i)=19mm). 5. The dependence of granulose synthase on a suitable polyglucan primer was demonstrated by using enzyme obtained from a granulose-free mutant strain (lacking ADP-glucose pyrophosphorylase). 6. Partial purification of granulose synthase from wild-type strains was facilitated by its being bound to the native particles of granulose. No activator was discovered, but ADP, AMP and pyridoxal 5'-phosphate were competitive inhibitors, ADP being most effective (K(i) about 0.2mm). 7. It would appear that the synthesis of granulose in Cl. pasteurianum is not subject to the positive, fine control that is a feature of glycogen biosynthesis in most bacteria.
D000244:Adenosine Diphosphate / Q000378:metabolism; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate / Q000378:metabolism; D001667:Binding, Competitive; D003013:Clostridium / Q000378:metabolism*; D005964:Glucosyltransferases / Q000302:isolation & purification / Q000378:metabolism; D009154:Mutation; D009243:NAD; D009249:NADP; D009713:Nucleotidyltransferases / Q000302:isolation & purification / Q000378:metabolism; D011134:Polysaccharides / Q000096:biosynthesis*; D011732:Pyridoxal Phosphate; D011773:Pyruvates
D011134:Polysaccharides; D011773:Pyruvates; D009243:NAD; D000249:Adenosine Monophosphate; D009249:NADP; C002819:granulose; D011732:Pyridoxal Phosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D005964:Glucosyltransferases; D009713:Nucleotidyltransferases