(CO2)-C-14 EXCHANGE WITH ACETOACETATE CATALYZED BY DIALYZED CELL-FREE-EXTRACTS OF THE BACTERIAL STRAIN BUNN GROWN WITH ACETONE AND NITRATE

The nitrate-reducing bacterial strain BunN is able to grow with acetone and nitrate under anoxic conditions. Dialyzed crude cell-free extracts of acetone-plus-nitrate-grown cells of strain BunN catalyzed the exchange of (CO2)-C-14 into acetoacetate in an ADP-dependent reaction. The rates of exchange catalyzed by extracts of acetate-grown or 3-hydroxybutyrate-grown cells were only 13% of that catalyzed by extracts of acetone-grown cells. The activity was enzymic since it was destroyed by boiling and was proper proportional to the amount of added extract. The optimal acetoacetate concentration was 100 mM and the apparent K-m was 11.1 mM. The pH optimum was 6.5, the exchange was not dependent on the addition of biotin, and the activity was not inhibited by avidin. The exchange activity was not stimulated (less than two fold) by a variety of metal ions or by a range of possible cofactors. Under optimal conditions (100 mM acetoacetate, 5 mM ADP, 10 mM NaHCO3, pH 6.5, under N-2), the exchange activity was 2.7 nmol . min(-1). mg protein(-1); 2% of the in vivo carboxylation activity of acetone-plus-nitrate-grown cultures. It is suggested that the exchange reaction is a partial reaction catalyzed by the enzyme (or enzyme complex) that carboxylates acetone, and that the methods developed in this study provide a means with which to investigate this reaction further.