DUAL ROLE OF DIVALENT-CATIONS IN THE BILE ACID-COA LIGASE CATALYZED REACTION

The role of divalent cations in the bile acid:CoA ligase catalyzed rea ction of cholic acid, CoA and ATP to yield cholyl-CoA was investigated using guinea pig liver microsomes as the source of enzyme. EDTA treat ment completely eliminated activity indicating an absolute requirement for divalent cation for enzyme activity. Analysis of this requirement revealed that it was twofold. First, the data suggested that ATP whic h was not complexed with a divalent cation did not appreciably bind to the enzyme and thus a divalent cation complex of ATP is the form of A TP that is the substrate for the enzyme. Further, this was shown to be the basis for the absolute requirement for divalent cation in the rea ction. In addition, analysis revealed that there is a secondary site w hich binds divalent cations with relatively low affinity, and results in a rate enhancement. Binding at this secondary site is estimated to increase the rate by greater than 60%.