Studies of the process of renaturation and assembly of Escherichia coli succinyl-CoA synthetase from its alpha and beta subunits.
Succinyl-CoA synthetase catalyzes the substrate-level phosphorylation step of the tricarboxylic acid cycle. The enzyme, as isolated from Escherichia coli, has an alpha 2 beta 2 subunit structure. It is known that substrate-binding sites are distributed between both subunit types and that the active enzyme is the nondissociating tetramer. This paper describes a study of the process of assembly of the enzyme from its denatured constituent subunits. Starting with equimolar mixtures of the subunits that are prepared in denaturing conditions (6 M urea, 5% acetic acid), rapid renaturation to produce virtually a fully active enzyme occurs after neutralization and dilution under suitable conditions. This process occurs most efficiently in the presence of either ATP or Pi, indicating that occupation of the phosphoryl-binding site on the refolding alpha subunit facilitates productive intrasubunit interactions. We have determined conditions of protein concentration, pH, temperature, final urea concentration, and buffer compositions that optimize both the rate and extent of production of active enzyme. The final refolded product is indistinguishable from the native species with respect to its specific catalytic activity, size, and other physical properties. To probe further the mechanism and route of renaturation, we have shown that the rate of appearance of activity has first-order dependence on each of the two subunits. The step that determines the rate of assembly is thus bimolecular, such as the association of structural monomers to form a dimeric transient species. The highly specific mutual interactions between the refolding transient species of subunits must be essential for the correct assembly of this enzyme from the two gene products in vivo.
Wolodko
W T
WT
Bridger
W A
WA
D000255:Adenosine Triphosphate; D002850:Chromatography, Gel; D003066:Coenzyme A Ligases / Q000032:analysis*; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium; D015636:Magnesium Chloride; D010710:Phosphates; D013389:Succinate-CoA Ligases / Q000032:analysis* / Q000037:antagonists & inhibitors; D013696:Temperature; D014508:Urea / Q000494:pharmacology
D046911:Macromolecular Substances; D010710:Phosphates; D015636:Magnesium Chloride; D000255:Adenosine Triphosphate; D014508:Urea; D003066:Coenzyme A Ligases; D013389:Succinate-CoA Ligases; D008274:Magnesium