Chemical modification of Escherichia coli succinyl-CoA synthetase with the adenine nucleotide analogue 5'-p-fluorosulphonylbenzoyladenosine.
Escherichia coli succinyl-CoA synthetase (EC 6.2.1.5) was irreversibly inactivated on incubation with the adenine nucleotide analogue 5'-p-fluorosulphonylbenzoyladenosine (5'-FSBA). Optimal inactivation by 5'-FSBA took place in 40% (v/v) dimethylformamide. ATP and ADP protected the enzyme against inactivation by 5'-FSBA, whereas desulpho-CoA, an analogue of CoA, did not. Inactivation of succinyl-CoA synthetase by 5'-FSBA resulted in total loss of almost four thiol groups per alpha beta-dimer, of which two groups appeared to be essential for catalytic activity. 5'-FSBA at the first instance appeared to interact non-specifically with non-essential thiol groups, followed by a more specific reaction with essential thiol groups in the ATP(ADP)-binding region. Plots of the data according to the method of Tsou [(1962) Sci. Sin. 11, 1535-1558] revealed that, of the two slower-reacting thiol groups, only one was essential for catalytic activity. When succinyl-CoA synthetase that had been totally inactivated by 5'-FSBA was unfolded in acidic urea and then refolded in the presence of 100 mM-dithiothreitol, 85% of the activity, in comparison with the appropriate control, was restored. These data are interpreted to indicate that inactivation of succinyl-CoA synthetase by 5'-FSBA involves the formation of a disulphide bond between two cysteine residues. Disulphide bond formation likely proceeds via a thiosulphonate intermediate between 5'-p-sulphonylbenzoyladenosine and one of the reactive thiol groups of the enzyme.
D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001665:Binding Sites; D003066:Coenzyme A Ligases / Q000037:antagonists & inhibitors*; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D008024:Ligands; D011487:Protein Conformation; D013389:Succinate-CoA Ligases / Q000037:antagonists & inhibitors*; D013438:Sulfhydryl Compounds
D008024:Ligands; D013438:Sulfhydryl Compounds; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D003066:Coenzyme A Ligases; D013389:Succinate-CoA Ligases; D000241:Adenosine; D004229:Dithiothreitol