Chemical modification of Escherichia coli succinyl-CoA synthetase with the adenine nucleotide analogue 5′-p-fluorosulphonylbenzoyladenosine

Abstract

E. coli succinyl-CoA synthetase (EC 6.2.1.5) was irreversibly inactivated on incubation with the adenine nucleotide analog 5''-p-fluorosulfonylbenzoyladenosine (5''-FSBA). Optimal inactivation by 5''-FSBA took place in 40% (vol/vol) dimethylformamide. ATP and ADP protected the enzyme against inactivation by 5''-FSBA, whereas desulfo-CoA, an analog of CoA, did not. Inactivation of succinyl-CoA synthetase by 5''-FSBA resulted in total loss of almost 4 thiol groups/.alpha..beta.-dimer, of which 2 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 revealed that, of the 2 slower-reacting thiol groups, only 1 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. Inactivation of succinyl-CoA synthetase by 5''-FSBA may involve the formation of a disulfide bond between 2 cysteine residues. Disulfide bond formation likely proceeds via a thiosulfonate intermediate between 5''-p-sulfonylbenzoyladenosine and 1 of the reactive thiol groups of the enzyme.