CHARACTERIZATION OF THE MONOVALENT AND DIVALENT-CATION REQUIREMENTS FOR THE XENOBIOTIC CARBOXYLIC-ACID - COA LIGASES OF BOVINE LIVER-MITOCHONDRIA

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 NH4+, Na+ only s upported a very low rate. Varying the nature of the anion had only a m inor effect. For XL-I and XI-II, the optimum concentration of K+ was 5 0 mM; higher (physiologic) concentrations led to a decrease in activit y. K+ did not inhibit XL-III. The absolute requirement for divalent ca tion was satisfied by Mg2+ or Mn2+, or to a lesser extent by Co2+ or F e2+. For the XL-I and XL-II, excess uncomplexed Mg2+ or Mn2+ decreased the rate; the optimum concentration of Mn2+ was approximately the sam e as the concentration of ATP in the assay, and the optimum concentrat ion of Mg2+ was approximately double the concentration of ATP in the a ssay. This is consistent with the concept that the divalent cation is required to complex with ATP and with the known stability constants fo r the ATP complexes of these two divalent cations. XL-III was not inhi bited 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 Mg2+ dependent, a nd that the cation effects are complex and differ for XL-I and XL-II a s compared with XL-III. (C) 1998 Elsevier Science B.V.