Characterization of the ATP- and GTP-specific Succinyl-CoA Synthetases in Pigeon

Abstract

Two succinyl-CoA synthetases, one highly specific for GTP/GDP and the other for ATP/ADP, have been purified to homogeneity from pigeon liver and breast muscle. The two enzymes are differentially distributed in pigeon, with only the GTP-specific enzyme detected in liver and the ATP-specific enzyme in breast muscle. Based on assays in the direction of CoA formation, the ratios of GTP-specific to ATP-specific activities in kidney, brain, and heart are ϳ7, 1, and 0.1, respectively. Both enzymes have the characteristic ␣-and ␤-subunits found in other succinyl-CoA synthetases. Studies of the ␣-subunit by electrophoresis, mass spectrometry, reversed-phase high performance liquid chromatography, and peptide mapping showed that it was the same in the two enzymes. Characterization of the ␤-subunits by the same methods indicated that they were different, with the tryptic peptide maps providing evidence that the ␤-subunits likely differ along their entire sequences. Because the two succinyl-CoA synthetases incorporate the same ␣-subunit, the determinants of nucleotide specificity must reside within the ␤-subunit. Determination of the apparent Michaelis constants showed that the affinity of the GTP-specific enzyme for GDP is greater than that of the ATP-specific enzyme for ADP (7 versus 250 M). Rather large differences in apparent K m values were also observed for succinate and phosphate.

Expression of Two Succinyl-CoA Synthetases with Different Nucleotide Specificities in Mammalian Tissues

“…The same ␣ subunit is found in both A-SCS and G-SCS enzymes (1). Northern blots revealed a single transcript of 1.5 kb with the strongest expression of ␣ observed in heart followed by kidney, liver, and testis.…”

“…Both enzymes are located in the matrix of mitochondria, where they could participate in the citric acid cycle. We previously found that G-SCS is by far the predominant enzyme detected by enzyme assay in pigeon liver, whereas A-SCS predominates in pigeon breast muscle (1).…”

“…We definitively showed by enzyme purification and characterization that GDP-forming and ADP-forming enzymes occur in pigeon liver and breast muscle, respectively (1). The two enzymes were found to contain the same ␣ subunit but different ␤ subunits that share 54% sequence identity (1,2).…”

Interaction between succinyl CoA synthetase and the heme-biosynthetic enzyme ALAS-E is disrupted in sideroblastic anemia

“…There are 2 isoforms of SCS, the GTP-specific (G-SCS) and ATP-specific isoforms (A-SCS) (33). Recently, it has been shown that the 2 isoforms of SCS contain the identical α subunit, although they differ in their β subunits, indicating that the β subunit determines the specificity of nucleotide binding (33,34).…”

Is There an Answer?

“…Knowledge of A-SCS in animals began with its purification from blowfly thorax (7), and its detection in tissue homogenates (8,9). We then purified A-SCS in pigeon breast muscle and G-SCS in liver, demonstrated that each enzyme is highly specific for its nucleotide, and showed that both contain alpha-and beta-type subunits (10). Although the same alpha subunit was found in both enzymes, the beta subunits were only 53% identical in sequence (11) and thus confer nucleotide specificity.…”