Biochemical characterization of a Rhizobium etli monovalent cation-stimulated acyl-coenzyme A carboxylase with a high substrate specificity constant for propionyl-coenzyme A

Abstract

Biotin has a profound effect on the metabolism of rhizobia. It is reported here that the activities of the biotin-dependent enzymes acetyl-coenzyme A carboxylase (ACC; EC 6.4.1.2) and propionyl-coenzyme A carboxylase (PCC; EC 6.4.1.3) are present in all species of the five genera comprising theRhizobiaceaewhich were examined. Evidence is presented that the ACC and PCC activities detectable inRhizobium etliextracts are catalysed by a single acyl-coenzyme A carboxylase. The enzyme fromR. etlistrain 12-53 was purified 478-fold and displayed its highest activity with propionyl-CoA as substrate, with apparentKmandVmaxvalues of 0·064 mM and 2885 nmol min−1(mg protein)−1, respectively. The enzyme carboxylated acetyl-CoA and butyryl-CoA with apparentKmvalues of 0·392 and 0·144 mM, respectively, andVmaxvalues of 423 and 268 nmol min−1(mg protein)−1, respectively. K+,or Cs+markedly activated the enzyme, which was essentially inactive in their absence. Electrophoretic analysis indicated that the acyl-CoA carboxylase was composed of a 74 kDa biotin-containingαsubunit and a 45 kDa biotin-freeβsubunit, and gel chromatography indicated a total molecular mass of 620 000 Da. The strong kinetic preference of the enzyme for propionyl-CoA is consistent with its participation in an anaplerotic pathway utilizing this substrate.