Dissociation and Association of Fumarase Subunits with Special Reference to the Formation of a Functional Tetramer

Swine heart fumarase is known to exist as a tetramer which can be dissociated with denaturing agents into inactive subunits. We have succeeded, for the first time, in reverting once‐dissociated subunits to form a functional tetramer, and studied the mechanism of the restoration of its activity. Incubation of fumarase in a medium containing 50 mM sodium phosphate buffer, pH 7.3, 10 mM dithiothreitol, 0.1 mM EDTA · 2 Na, 10% glycerol, 200 mM KCl and 6 M urea at 25°C for 30 min resulted in dissociation of the tetrameric enzyme into monomeric subunits which were totally inactive and lost most α‐helical structure, with all sulfhydryl groups freely accessible to the medium. The denatured subunits could then be reassociated by dialysis at 4°C for 24 h against the same medium as above with omission of 6 M urea. The product was found to be an inactive dimer which could be transformed into an enzymatically active tetramer only after incubation at 25°C for 3 h. The final product was indistinguishable from the native fumarase in physical, chemical and enzymatic properties. All the ingredients of the media were found to be necassary to achieve freely reversible and complete conversion of monomer to tetramer. An intermediate product, a dimer, showed fluorescence and circular dichroism spectra almost indistinguishable from those of a tetramer, but it was highly susceptible to tryptic digestion while the tetramer was strongly resistant. It is concluded that a small but very important conformational change associates with the conversion of dimers to tetramers, finalizing proper orientation of the active site residues.