Assay, purification and characterization of cob(I)alamin adenosyltransferase of Protaminobacter ruber

A simple spectrophotometric method for assay of ATP: cob(I)alamin Co-beta-adenosyltransferase was established. By using this method, specific activity of the enzyme in Protaminobacter ruber was found to be the highest in the logarithmic growth phase. The enzyme was highly purified from the cells in that phase and characterized. The molecular weight of the enzyme was estimated as 44,000 by gel filtration. The optimal pH and temperature were about 8.0 and 50 degrees C, respectively. Among divalent metal ions tested, only Mg2+ was effective for the enzyme activity. Km values for ATP and hydroxocobalamin were 250 microM and 26.3 microM, respectively. Substrate analogs such as GTP and CTP had only small activity in the enzymatic reaction.