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 expr ession, the enzyme was purified from the E. coli in a three-step proce ss consisting of (NH4)(2)SO4 precipitation, gel filtration and anion-e xchange chromatography. The purified enzyme gave one band on gel elect rophoresis and was judged essentially free of impurities, although it was unstable when diluted without the addition of 15 mu M BSA. The iso electric 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 f or 10 min at pH 3.0 or at pH 12.4. When isolated, the recombinant enzy me was fully active without the addition of extra Mg2+. The K-m for gl ucose 1-phosphate was much higher than that of other PGM species repor ted, 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 kinet ic constants were determined: V-max 81.1 units/mg; k(cat) and the turn over rate 135 s(-1); K-m (glucose 1,6-diphosphate) 0.2 mu M; K-m (gluc ose 1-phosphate) 2.6 mM; k(cat)/K-m (glucose 1-phosphate) 5.2 x 10(4) M-1 . s(-1). The recombinant enzyme is considered to follow a characte ristic substituted enzyme or Ping Pong reaction mechanism.