PURIFICATION AND CHARACTERIZATION OF ASCORBIC-ACID PHOSPHORYLATING ENZYME FROM PSEUDOMONAS-AZOTOCOLLIGANS

A enzyme that catalyzed the specific formation of ascorbic acid-2-phosphate (AsA2P) from ascorbic acid (AsA) and adenosine-5'-triphosphate (ATP), was purified 3,200-fold to homogeneity from a cell extract of Pseudomonas azotocolligans. The purified enzyme appeared as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and consisted of a single polypeptide with a molecular weight of about 30,000. Of phosphoryl donors tested, p-nitrophenylphosphate (p-NPP) and pyrophosphate (PPi) were as effective as ATP. Optimal pHs for the phosphorylating activity were around 4.0 and 5.5 when PPi and ATP were used as phosphoryl donors, respectively. The Km for AsA was 147 mM. The enzyme activity was inhibited by Cu2+, but not by sulfhydryl reagents. The enzyme simultaneously had phosphatase activity at weakly acidic or neutral pH and the Km for p-NPP in the phosphatase activity was 0.38 mM. The enzyme was tentatively named "ascorbic acid phosphorylating enzyme."