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

Succinyl-coenzyme A synthetase [succinate:CoA ligase (ADP-forming), EC 6.2.1.5] of Escherichia coli in an alpha 2 beta 2 tetramer. A histidyl residue in the alpha subunit is phosphorylated as a catalytic intermediate. It has been suggested [Bild, G. S., Janson, C. & Boyer, P. D. (1980) J. Biol. Chem. 255, 8109--8115] that the mechanism of action of this enzyme involves intersubunit cooperativity in which attachment of substrates at one of the two active sites promotes catalytic events at the other. This scheme would require that the two active sites, although otherwise equivalent, should act alternately. We have prepared a hybrid enzyme species that contains one 35S-labeled alpha subunit (dephosphorylated), one nonradioactive alpha subunit (phosphorylated), and two beta subunits per tetrameric molecule. With the aid of a selective chromatographic procedure for the isolation of peptides that contain phosphohistidyl residues, we have shown that each of the alpha subunits undergoes phosphorylation when the hybrid enzyme is exposed briefly to substrates. This result demonstrates that the two active sites are capable of alternate activity and lends 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 two alpha subunits.