Reversible pH-induced dissociation of glucose dehydrogenase from Bacillus megaterium. I. Conformational and functional changes

Abstract

Glucose dehydrogenase from Bacillus megaterium exists as a stable tetramer at pH 6.5, but is readily dissociated into four enzymatically inactive protomers by simply raising the pH to 9. Readjustment to the original pH value, however, effects a complete reassociation and reactivation of the enzyme. The conformational and functional changes during dissociation were studied using various spectroscopic methods: due to the exposure of hitherto buried tryptophan residues to the solvent medium, ultraviolet absorption, circular dichroism and fluorescence spectra are in part changed drastically upon dissociation. Light scattering measurements show that the exposure of the tryptophan moieties, and likewise the enzymic inactivation, are not strictly concomitant with the degradation of quaternary structure. This result suggests a partial inactivation, combined with a rearrangement of the protein structure, before the actual dissociation step. The circular dichroism of the peptide backbone reveals only slight differences between the secondary structure of the dissociated enzyme and the native tetramer. All conformation and functional changes in the course of the dissociation must be fully reversible, since the spectroscopic and enzymatic properties of the native and a tetrameric enzyme, reassociated from its subunits, are absolutely identical. Chloride and phosphate ions stabilize the tetrameric state of glucose dehydrogenase, and thus strongly influence both rate and extent of dissociation and reassociation. Circular dichroism measurements show this stabilization to be correlated with an ion-induced tightening of the protein structure.