Chemical Modification of Tryptophan Residues in Escherichia coli Succinyl-CoA Synthetase. Effect on Structure and Enzyme Activity

Succinyl-CoA synthetase of Escherichia coli is an α₂β₂ protein containing active sites at the interfaces between a- and β-subunits. The a-subunit contains a histidine residue that is phosphorylated during the reaction. The β-subunit binds coenzyme A and probably succinate [see Nishimura, J. S. (1986) Adv. Enzymol. Relat. Areas Mol. Biol. 58, 141–172]. Chemical modification studies have been conducted in order to more clearly define functions of each subunit. Tryptophan residues of the enzyme were modified by treatment with N-bromosuccinimide at pH 7. There was a linear relationship between loss of enzyme activity and tryptophan modified. At one tryptophan residue modified per β-subunit, 100% of the enzyme activity was lost. In this enzyme sample, one methionine residue in each α- and β-subunit was oxidized to methionine sulfoxide, although loss of enzyme activity could not be related in a linear manner to the formation of this residue. Subunits were prepared from enzyme that was inactivated 50% by N-bromosuccinimide with 0.5 tryptophan modified per β-subunit but with insignificant modification of methionine residues in either subunit. Small decreases in the tyrosine and histidine content were observed in the a-subunit but not in the β-subunit. In this case, modified β-subunit when mixed with unmodified a-subunit gave a population of molecules that was 50% as active as the refolded, unmodified control but was only slightly changed with respect to phosphorylation capacity and unchanged with respect to rate of phosphorylation. Relatively slight changes were observed in the phosphorylation capacity of the a-subunit both alone or when refolded with β-subunit. The results suggest that modification of a tryptophan residue in the β-subunit causes loss of catalytic activity but does not interfere with its ability to refold with the a-subunit to establish a conformation of the latter that is similar to that in the native enzyme. Failure of substrates and the CoA analogue desulfo-CoA to protect the enzyme against inactivation by N-bromosuccinimide would appear to indicate that the modified tryptophan residue is not at the active site.