A phosphorus 31 nuclear magnetic resonance study of the intermediates of the Escherichia coli succinyl coenzyme A synthetase reaction. Evidence for substrate synergism and catalytic cooperativity.

Escherichia coli succinyl-CoA synthetase has an a& structure (molecular weight, 140,000) with the two active sites arranged at the interface of the a and j3 subunits.Here we describe 31P-NMR experiments confirming the existence of two phosphorylated intermediates in catalysis.The phosphohistidyl resonance is readily observed at -4.8 ppm.This resonance is shifted upfield in the presence of M&+ and broadened by Mn", indicating interaction between the metal ion and the phosphoryl group.The addition of succinate alone is without effect on the spectrum.The presence of CoA causes a downfield shift and broadening of the phosphohistidyl resonance to a line width sufficiently large to be indicative of two exchanging conformations.The presence of both CoA and the competitive inhibitor 2,2'difluorosuccinate seems to freeze the phosphoryl group in one orientation.The addition of ATP to this already phosphorylated enzyme leads to appearance of a succinyl-phosphate resonance.This last result is in harmony with the concept of alternating sites catalytic cooperativity previously proposed for this enzyme, since it implies that ATP binding or phosphorylation of histidine at one active site triggers phosphoryl transfer from histidine to succinate at the other site.If the nonhydrolyzable P,y-methylene analogue of ATP is used, however, this produces no change in the phosphohistidyl resonance.This suggests that intrasubunit communication is triggered by phosphorylation by ATP rather than by binding of nucleotide.We also observed CoA-induced changes in the spectrum of the enzyme-bound j3-y-methylene analogue of ATP, consistent with the previously observed CoA-mediated enhancement of the ATP -+ ADP exchange which is known as "substrate synergism."A detailed model €or catalysis consistent with these observations is presented.Succinyl-CoA synthetase from E. coli is a tetrameric enzyme with an ~$ 3 ~ structure and an overall molecular weight