Studies on recombinant Acetobacter xylinum alpha-phosphoglucomutase.
The phosphoglucomutase (PGM) from Acetobacter xylinum, which had been cloned and expressed in Escherichia coli, has been studied. After expression, the enzyme was purified from the E. coli in a three-step process consisting of (NH4)2SO4 precipitation, gel filtration and anion-exchange chromatography. The purified enzyme gave one band on gel electrophoresis and was judged essentially free of impurities, although it was unstable when diluted without the addition of 15 microM BSA. The isoelectric point for A. xylinum PGM was 4.8 and the molar absorbance was 3.9 x 10(4) M-1.cm-1. The enzyme was reasonably heat-stable below 50 degrees C and was stable throughout the pH 5.5-7.4 range, but was 70% inactivated at pH 10.0 and completely inactivated after standing for 10 min at pH 3.0 or at pH 12.4. When isolated, the recombinant enzyme was fully active without the addition of extra Mg2+. The Km for glucose 1-phosphate was much higher than that of other PGM species reported, which accords with the production of extracellular cellulose in A. xylinum. Glucose 1,6-diphosphate is not considered to be a substrate or coenzyme but an activating cofactor like Mg2+. The following kinetic constants were determined: Vmax 81.1 units/mg; kcat and the turnover rate 135 s-1; Km (glucose 1,6-diphosphate) 0.2 microM; Km (glucose 1-phosphate) 2.6 mM; kcat/Km (glucose 1-phosphate) 5.2 x 10(4) M-1.s-1. The recombinant enzyme is considered to follow a characteristic substituted enzyme or Ping Pong reaction mechanism.
Kvam
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SINTEF, Applied Chemistry, Blindern, Oslo, Norway.
Olsvik
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McKinley-McKee
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Saether
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D000838:Anions; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004795:Enzyme Stability / Q000187:drug effects; D041822:Gluconacetobacter xylinus / Q000201:enzymology* / Q000235:genetics; D005990:Glycerol / Q000494:pharmacology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D010733:Phosphoglucomutase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D014325:Tromethamine; D015032:Zinc / Q000378:metabolism
D000838:Anions; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D014325:Tromethamine; D012710:Serum Albumin, Bovine; D010733:Phosphoglucomutase; D008274:Magnesium; D015032:Zinc; D005990:Glycerol