Characterization of the ATP- and GTP-specific succinyl-CoA synthetases in pigeon. The enzymes incorporate the same alpha-subunit.
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 approximately 7, 1, and 0.1, respectively. Both enzymes have the characteristic alpha- and beta-subunits found in other succinyl-CoA synthetases. Studies of the alpha-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 beta-subunits by the same methods indicated that they were different, with the tryptic peptide maps providing evidence that the beta-subunits likely differ along their entire sequences. Because the two succinyl-CoA synthetases incorporate the same alpha-subunit, the determinants of nucleotide specificity must reside within the beta-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 microM). Rather large differences in apparent Km values were also observed for succinate and phosphate.
Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota 58202, USA.
J D
JD
Johnson
W W
WW
Muhonen
D O
DO
Lambeth
D000214:Acyl Coenzyme A / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D010856:Columbidae; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009132:Muscles / Q000201:enzymology*; D010449:Peptide Mapping; D011487:Protein Conformation; D013379:Substrate Specificity; D013389:Succinate-CoA Ligases / Q000302:isolation & purification* / Q000378:metabolism; D019802:Succinic Acid / Q000378:metabolism
D000214:Acyl Coenzyme A; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D019802:Succinic Acid; C012046:succinyl-coenzyme A; D013389:Succinate-CoA Ligases