Isolation of the alpha and beta subunits of Escherichia coli succinyl coenzyme A synthetase and their recombination into active enzyme.

Pure preparations of the alpha and beta subunits of succinyl-CoA synthetase of Escherichia coli have been obtained by preparative gel electrophoresis in the presence of detergent or by gel filtration in acid urea solution. Substantial enzyme activity (50 to 60%) can be recovered following renaturation of an equimolar mixture of the two subunits prepared by gel filtration. The substrate ATP is required for reconstitution of activity, but the presence of our substrates does not appear to influence the renaturation process. Knowing that ATP phosphorylates the alpha subunit in the native enzyme, these data suggest that phosphorylation of this subunit is necessary for correct assembly. Comparison of the amino acid compositions of the stoichiometry of the subunit reassociation in the reconstitution process confirm the alpha2 beta2 structure for the native enzyme.