Abstract:
Escherichia coli citrate synthase (CS) is a hexamer of identical subunits which is allosterically inhibited by NADH. Although its three-dimensional structure has not yet been determined, a satisfactory model of the subunit fold can be constructed using the homologous vertebrate CS, a dimer whose structure is known, as a guide. Equilibrium unfolding of E. coli CS by chaotropic agents or heat, whether probed by circular dichroism or by fluorescence, occurs in two stages, with an intermediate (I state) that is stable between 2.5 and 5.5 M urea, approximately. The two transitions are associated with 67% and 33% loss of structure, respectively. Only the second transition is reversible. Urea-gradient gel electrophoresis and electrospray ionization-time-of-flight mass spectrometry (ESI-TOF MS) were used to show that the I state is a collection of aggregates. Acinetobacter anitratum CS, also allosteric and hexameric, unfolds via one transition, unlike E. coli CS. Unfolding exper ments with CS chimeras, in which onestructural domain came from E. coli CS and the other from A. anifratum CS, suggested that the existence of an I state is associated with an E. coli large domain. Cavity-creating mutations were introduced into the cores of the two domains of E. coli CS. The mutant proteins also showed biphasic unfolding. Some mutants exhibited a stabilized first transition, a behaviour that can be explained by destabilization of the I state. The secondary structure features in which these mutations are located form the cores of the two domains. A model of the I state constructed from these results shows a mostly hydrophobic surface, consistent with the tendency of the intermediate to aggregate. ESI-TOF MS was used to study the quaternary structure of CS and also CS-ligand interactions. CS exhibits an equilibrium between dimers and hexamers that is pH and protein-concentration dependent. The association constant (K$\sb{\rm A}$) for the dimer-hexamer equilibrium was determined, and it was shown that NADH binds strongly only to hexamer and converts dimers to hexamers. The dissociation constants (K$\sb{\rm D}$) for NADH binding to dimer and hexamer were calculated from the mass spectrometric data. This study establishes a relationship between quaternary structure and NADH binding in E. coli CS.
