A dimeric form of Escherichia coli succinyl-CoA synthetase produced by site-directed mutagenesis

Succinyl-CoA synthetase (SCS) catalyzes the substrate-level phosphorylation step of the citric acid cycle. The enzyme from Escherichia coli is an (alp ha beta)(2)-heterotetramer with two active sites, one in each alpha beta-di mer. To determine whether the two active sites could function independently , mutations were made to split the tetramer into alpha beta-dimers. Because two choices for the tetramer (I and II) were possible from the X-ray cryst allographic analyses, mutations were made at two different interfaces. All mutations based on tetramer I resulted in an intact tetramer. Of the two mu tants based on tetramer II, one was insoluble and the other, where M156 bet a, Y158 beta, R161 beta and E162 beta were changed to D, D, E and R, respec tively, was a dimer. This quaternary structure was confirmed by fast protei n liquid chromatography, blue native PAGE and ultracentrifugation. The DDER mutant has kinetic parameters similar to the tetrameric E. coli enzyme. Li ke the tetrameric enzyme, it shows ATP-facilitated dethiophosphorylation, p roving that this property is a single-site effect. The ATP-facilitated deth iophosphorylation is inhibited by phosphate. It is concluded that dimerizat ion of ap-dimers is not a prerequisite for-catalytic competency nor for alt ernating sites cooperativity in the tetramer. The rationale behind the dime r-of-dimers in E. coli SCS is still not known, but increased solubility, in creased stability and in vivo interactions of the tetramer with other prote ins are still possibilities. (C) 1999 Academic Press.