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

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 typical of noninteracting globular proteins with no evidence of size heterogeneity. The sedimentation coefficients at infinite dilution (s20,w) 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.(ABSTRACT TRUNCATED AT 250 WORDS)