Chemical modification and hybrid enzyme formation as probes of the active site and subunit interactions in Escherichia coli succinyl-CoA synthetase.
Succinyl-CoA synthetase from Escherichia coli has an alpha 2 beta 2 subunit structure and is known to display half-of-the-sites reactivity with respect to its phosphorylation by ATP. The studies reported herein are a component of our attempts to rationalize the heterologous tetrameric structure in terms of catalytic function. The isolated refolded beta subunit interacts specifically with an affinity column of agarose-hexane-CoA, consistent with the idea that the CoA-binding subsite of the active center is located on the beta subunit. The enzyme is inactivated by phenylglyoxal according to biphasic kinetics; saturating levels of the substrates CoA and ATP, alone or in combination, give only partial protection against such inactivation. Treatment of the enzyme with the sulfhydryl reagent 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) resulted in rapid inactivation, accompanied by the reaction of three to four-SH groups per molecule; prolonged incubation with NBD-Cl eventually results in reaction of 16 of the 24 sulfhydryl groups of the tetramer. Hybrid enzyme preparations have been constructed by refolding mixtures containing beta and various ratios of native and NBD-Cl-modified alpha subunits. The loss of activity associated with the incorporation of chemically modified alpha is not that predicted by a simple model based on binomial distribution, but is complex and consistent with the kind of intersubunit communication that may be expected for catalytic cooperativity between the two active sites of the enzyme molecule.
D009327:4-Chloro-7-nitrobenzofurazan; D001120:Arginine; D001665:Binding Sites; D003066:Coenzyme A Ligases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D011487:Protein Conformation; D013389:Succinate-CoA Ligases / Q000378:metabolism*
D001120:Arginine; D003066:Coenzyme A Ligases; D013389:Succinate-CoA Ligases; D009327:4-Chloro-7-nitrobenzofurazan