Active enzyme sedimentation, sedimentation velocity, and sedimentation equilibrium studies of succinyl-CoA synthetases of porcine heart and Escherichia coli

Abstract

Succinyl-CoA synthetases from Escherichia coli and porcine heart muscle have been viewed as prototypes of two classes of the enzyme. The bacterial enzyme has been reported to be an .alpha.2.beta.2 tetramer, with many suggestions in the literature for cooperative interactions between active sites that may contribute to its catalytic efficacy. In contrast, gel filtration experiments of others have indicated that the heart enzyme is a simple .alpha..beta. dimer, with no evidence of dimerization or interaction between like sites. All previous estimates of molecular size of these enzymes have been carried out at concentrations that are much higher than those that are used during activity measurements. The present study was carried out to confirm the differences in the quaternary structures of these two species of succinyl-CoA synthetase and to extend our knowledge of these structures to very low concentrations to enable correlation of their subunit structures with their catalytic properties. Conventional sedimentation velocity centrifugation with both enzymes indicates behavior tyhpical of noninteracting globular proteins with no evidence of size heterogeneity. The sedimentation coefficients at infinite dilution .**GRAPHIC**. have been determined to be 7.04 S and 4.55 S for the E. coli and porcine heart enzymes, respectively. Sedimentation velocity measurements have been extended to very low enzyme concentrations (typical of those used in activity measurements) by active enzyme centrifugation experiments, in which we have determined the rate of sedimentation of a zone of active enzyme through a chromogenic substrate solution. These measurements confirm that the differences in sedimentation coefficients extend to assay concentration and give no indication of dissociation of the tetrameric E. coli enzyme nor association of the dimeric porcine heart enzyme. Sedimentation equilibrium experiments have been performed for both enzymes over a wide range of loading concentrations. Consistent with observations made with previous sedimentation equilibrium runs, E. coli succinyl-CoA synthetase behaves anomalously at low concentrations (< 2 mg/mL), indicating the presence of one or more components that are less than the tetramer in size. The sedimentation equilibrium data exclude a simple dissociation-equilibrium system, however, and the active enzyme centrifugation results with the same enzyme show that the smaller components, whatever their nature, have no significant activity. At higher concentrations of E. coli succinyl-CoA synthetase where the anomalous behavior is not observed, the apparent molecular weight is concentration-independent and was determined to be 13900, in good agreement with the value of 142000 that has been calculated from its amino acid sequence and an .alpha.2.beta.2 structure [Buck, D., Spencer, M. E., and Guest, J. R. (1985) Biochemistry 24, 6245-6252]. Thus, appropriate correlation of molecular weight to sedimentation coefficient confirms that the active species is the tetramer. The porcine heart enzyme, in contrast, has a Mr of 78,000, with no detectable formation of larger aggregates even at concentrations in excess of 10 mg/mL, and reduced tendency to form anomalous smaller components. Thus, correlation of all of our sedimentation data obtained over a wide concentration range (ca. 1 .mu.g/mL to 10 mg/mL) establishes that the active form of E. coli succinyl-CoA synthetase is a nondissociating .alpha.2.beta.2 tetramer while the porcine heart species of enzyme is a nonassociating .alpha..beta. dimer.