Characterization of the monovalent and divalent cation requirements for the xenobiotic carboxylic acid: CoA ligases of bovine liver mitochondria

Abstract

The XL-I, XL-II and XL-III forms of xenobiotic/medium-chain fatty acid: CoA ligase were found to be inactive toward benzoate in the absence of either monovalent or divalent cations. The absolute requirement for monovalent cation was satisfied by either K +, Rb +, or NH + 4. Na + only supported a very low rate. Varying the nature of the anion had only a minor effect. For XL-I and XI-II, the optimum concentration of K + was 50 mM; higher (physiologic) concentrations led to a decrease in activity. K + did not inhibit XL-III. The absolute requirement for divalent cation was satisfied by Mg 2+ or Mn 2+, or to a lesser extent by Co 2+ or Fe 2+. For the XL-I and XL-II, excess uncomplexed Mg 2+ or Mn 2+ decreased the rate; the optimum concentration of Mn 2+ was approximately the same as the concentration of ATP in the assay, and the optimum concentration of Mg 2+ was approximately double the concentration of ATP in the assay. This is consistent with the concept that the divalent cation is required to complex with ATP and with the known stability constants for the ATP complexes of these two divalent cations. XL-III was not inhibited by uncomplexed divalent cations. Uncomplexed ATP was a moderate inhibitor of XL-I and XL-II, and a weak inhibitor of XL-III. The data indicate that in vivo benzoate conjugation is K + and Mg 2+ dependent, and that the cation effects are complex and differ for XL-I and XL-II as compared with XL-III.