Cloning, characterization, and expression of the β subunit of pig heart succinyl‐CoA synthetase

Abstract

The form of succinyl‐CoA synthetase found in mammalian mitochondria is known to be an αβ dimer. Both GTP‐and ATP‐specific isozymes are present in various tissues. We have isolated essentially identical complementary DNA clones encoding the β subunit of pig heart succinyl‐CoA synthetase from both newborn and adult tissues. These cDNAs include a 1.4‐kb sequence encoding the cytoplasmic precursor to the β subunit comprised of 417 amino acid residues including a 22‐residue mitochondrial targeting sequence. The cDNA encoding the 395‐amino acid, 42,502‐Da mature protein was confirmed to be the succinyl‐CoA synthetase β subunit by agreement with the N‐terminal protein sequence and by high homology to prokaryotic forms of the β subunit that were previously cloned (about 45% identical to β from Escherichia coli). In contrast to a previous report (Nishimura, J.S., Ybarra, J., Mitchell, T., & Horowitz, P.M., 1988, Biochem. J. 250, 429–434), we found no tryptophan residue to be encoded in the sequence for the mature β subunit, and this finding is corroborated by the fact that highly purified pig heart succinyl‐CoA synthetase shows no tryptophan fluorescence or tryptophan content in amino acid compositional analysis. The cDNA clones encoding the mature pig heart β subunit and its counterpart α subunit were coexpressed in a deletion mutant strain of E. coli. Recovery of succinyl‐CoA synthetase activity demonstrated that this combination of subunits forms a productive enzymatic complex having GTP specificity.