Purification and properties of the synthetase catalyzing the biotination of the aposubunit of transcarboxylase from Propionibacterium shermanii.

The synthetase that attaches biotin to the aposubunit of transcarboxylase (biotin-[methylmalonyl-CoA-carboxyltransferase]ligase) (EC 6.3.4.9) was purified to homogeneity by ion-exchange chromatography on cellulose DE-52 and CM-cellulose. The synthetase is a monomer of molecular weight 30,000. The pH and temperature optima for the synthetase are 6.0 and 37 degrees C, respectively. The apparent Km for the substrates ATP, biotin, and apo 1.3 S subunit of apotranscarboxylase are 38, 2.0, and 0.9 microM, respectively. Ni2+, Co2+, Zn2+, or Mn2+ could replace Mg2+ in the reaction. The affinity of synthetase toward metals is as follows: Zn2+ greater than Ni2+ greater than Mn2+ greater than Co2+ greater than Mg2+, and the activity with Zn2+ was much greater than that with the other divalent metals. EDTA completely inactivates the enzyme. The metals are necessary not only for the catalytic activity but also for the storage stability of the enzyme. The synthetase shows absolute specificity toward ATP.