CLONING, CHARACTERIZATION, AND EXPRESSION OF THE BETA-SUBUNIT OF PIG-HEART SUCCINYL-COA SYNTHETASE

The form of succinyl-CoA synthetase found in mammalian mitochondria is known to be an alphabeta dimer. Both GTP- and ATP-specific isozymes a re present in various tissues. We have isolated essentially identical complementary DNA clones encoding the beta subunit of pig heart succin yl-CoA synthetase from both newborn and adult tissues. These cDNAs inc lude a 1.4-kb sequence encoding the cytoplasmic precursor to the beta subunit comprised of 417 amino acid residues including a 22-residue mi tochondrial targeting sequence. The cDNA encoding the 395-amino acid, 42,502-Da mature protein was confirmed to be the succinyl-CoA syntheta se beta subunit by agreement with the N-terminal protein sequence and by high homology to prokaryotic forms of the beta subunit that were pr eviously cloned (about 45% identical to beta from Escherichia coli). I n contrast to a previous report (Nishimura, J.S., Ybarra, J., Mitchell , T., & Horowitz, P.M., 1988, Biochem. J. 250, 429-434), we found no t ryptophan residue to be encoded in the sequence for the mature beta su bunit, and this finding is corroborated by the fact that highly purifi ed pig heart succinyl-CoA synthetase shows no tryptophan fluorescence or tryptophan content in amino acid compositional analysis. The cDNA c lones encoding the mature pig heart beta subunit and its counterpart a lpha subunit were coexpressed in a deletion mutant strain of E. coli. Recovery of succinyl-CoA synthetase activity demonstrated that this co mbination of subunits forms a productive enzymatic complex having GTP specificity.