ENOLASE FROM BACILLUS STEAROTHERMOPHILUS

Enolase from Bacillus stearothermophilus was homogeneous in the ultracentrifuge and had the hydrodynamic properties of a globular protein with a molecular weight of 3.51 times 105 (sedimentation-diffusion) and 3.60 times 105 (Archibald approach to equilibrium). Electrophoretic and gel filtration measurements in presence of dissociating agents indicated that the enzyme consists of eight apparently identical polypeptide chains of molecular weight of 47 000. The denaturation of enolase has been studied as a function of urea concentration. A broad transition curve was observed, the enzyme in 8 M urea being inactive and dissociated into monomers. The far-ultraviolet circular dichroism spectrum of enolase in 8 M urea solution indicates extensive unfolding of the globular structure. Accordingly, fluorescence emission measurements indicate that the tryptophanyl residues of the denatured enolase are largely exposed to the aqueous solvent medium. The enzyme can be reversibly denatured by 8 M urea and the enzymatic activity then recovered by dilution 1:100 of the denatured enzyme into buffer or by removing urea by dialysis. Under suitable conditions and in the presence of Mg2+-ions, 90–100% of original activity was recovered and the renatured enzyme was indistinguishable from the native one, as judged from several enzymological and physicochemical criteria, including molecular weight. The far-ultraviolet circular dichroism spectrum and the fluorescence emission properties of the native enzyme were regained very rapidly (less than 1 min), while enzymatic activity was recovered much more slowly. Octameric enzyme species were obtained on refolding the denatured enzyme at temperatures between 4 and 55.