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.
Johnson
J D
JD
Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota 58202, USA.
Muhonen
W W
WW
Lambeth
D O
DO
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