Role of Cobalt in Stabilizing the Molecular Structure of Glucose Isomerase fromStreptomyces griseofuscusS-41

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Abstract
The role of cobalt in the stabilization of the molecular structure of glucose isomerase from Streptomyces griseofuscus S-41 was investigated using various denaturants. The enzymatic activity and molar ellipticity at 220 nm were significantly reduced in 8 m urea solution, but restored to the original values on removing urea. Therefore, the enzyme was thought not to suffer a drastic conformational change with urea. On the other hand, the destruction of ordered structure involving a complete loss of activity was observed from circular dichroism and fluorescence spectra in 6 M guanidine hydrochloride solution. The enzyme showed somewhat peculiar behavior in organic solvents; in linear-chained solvents the activity correspondingly decreased with solvent concentrations, whereas it increased slightly in side-chained solvents and acetone. The change in activity observed here were not reflected in the circular dichroism and fluorescence spectra. Three of four cobalt ions originally contained in the enzyme were eliminated by treatment with EDTA or 8 M urea without significant loss of activity. However, a cobalt-free enzyme was quite difficult to obtain in stable form. For elimination of all cobalts, drastic treatment was required, such as with 6 M guanidine hydrochloride, acid-8M urea or EDTA-8M urea, indicating considerable dissociation into subunits. The cobalt addition showed a protective effect on the enzyme from denaturation in such drastic conditions, whereas it did not in less drastic conditions, with 8 M urea or organic solvents. It was, therefore, considered that one of the four cobalts was tightly bound to the enzyme and had an essential role in holding the ordered conformation, especially the quaternary structure of the enzyme, while the other three were bound loosely and might be less important in stabilizing the structure.

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