Capacity for alternating sites cooperativity in catalysis by succinyl-coenzyme A synthetase.

Abstract

Succinyl CoA [coenzyme A] synthetase [succinate:CoA ligase (ADP-forming), EC 6.2.1.5] of Escherichia coli is an .alpha.2.beta.2 tetramer. A histidyl residue in the .alpha. subunit is phosphorylated as a catalytic intermediate. It was suggested that the mechanism of the action of this enzyme involves intersubunit cooperativity in which attachment of substrates at 1 of the 2 active sites promotes catalytic events at the other. This scheme would require that the 2 active sites, although otherwise equivalent, should act alternatively. A hybrid enzyme species that contains 1 35S-labeled .alpha. subunit (dephosphorylated), 1 nonradioactive .alpha. subunit (phosphorylated) and 2 .beta. subunits/tetrameric molecule was prepared. With the aid of a selective chromatographic procedure for the isolation of peptides that contain phosphohistidyl residues, it was shown that each of the .alpha. subunits undergoes phosphorylation when the hybrid enzyme is exposed briefly to substrates. The 2 active sites are evidently capable of alternate activity, lending support to the concept of alternating sites cooperativity. The half-of-the-sites phosphorylation that occurs with this enzyme is not a consequence of permanent asymmetry or other lack of equivalence of the 2 .alpha. subunits.