Phosphorylation and formation of hybrid enzyme species test the "half of sites" reactivity of Escherichia coli succinyl-CoA synthetase

Recent sequencing experiments have identified alpha-His246 as the phosphorylation site of Escherichia coli succinyl-CoA synthetase Buck, D., Spencer, M. E., & Guest, J. R. (1985) Biochemistry 24, 6245-6252]. We have replaced alpha-His246 with an asparagine residue using site-directed mutagenesis techniques. The resulting mutant enzyme (designated H246N) exhibited no enzyme activity, as expected, but was found as a structurally intact, stable tetramer. Small differences in the net charge of H246N and wild-type enzymes were first detected on native polyacrylamide gels. These charge differences were resolved by using native isoelectric focusing gels to further separate the wild-type enzyme into diphosphorylated, monophosphorylated, and unphosphorylated species. The enzyme species were found to be interconvertible upon incubation with the appropriate enzyme substrate(s). Sample mixtures containing increasing molar ratios of H246N (alpha H246N beta)2 to wild-type enzyme (alpha beta)2 were unfolded and then refolded. The refolded enzyme mixtures were analyzed for enzymatic activity and separated on native isoelectric focusing gels. The hybrid enzyme (alpha beta alpha H246N beta) retained a significant amount of enzyme activity and also exhibited substrate synergism (stimulation of succinate in equilibrium succinyl-CoA exchange in the presence of ATP). Substrate synergism with this enzyme has been interpreted as evidence for interaction between active sites in such a way that only a single phosphoryl group is covalently attached to the enzyme at a given time Wolodko, W. T., Brownie, E.R., O'Connor, M. D., & Bridger, W. A. (1983) J. Biol. Chem. 258, 14116-14119]. On the contrary, we conclude that tetrameric succinyl-CoA synthetase from E. coli is comprised of two independently active dimer molecules associated together to form a "dimer of dimers" that displays substrate synergism within each dimer and not necessarily between dimers.(ABSTRACT TRUNCATED AT 250 WORDS)