Purification and Properties of Glycerol Dehydrase

Glycerol dehydrase, a coenzyme B12-dependent enzyme, from Aerobacter aerogenes, has been purified to a stable, inactive, homogeneous complex with hydroxocobalamin. The inactive complex can be converted to the active holoenzyme by replacement of hydroxocobalamin with coenzyme B12 in the presence of Mg++ and SO3=. The molecular weight of the hydroxocobalamin apoenzyme complex is 188,000 and 1 mole of hydroxocobalamin is bound to 1 mole of the apoenzyme. There is a significant difference between the spectra of the dissociated and the protein-bound hydroxocobalamin. The greater stability of the protein hydroxocobalamin complex as compared to the apoenzyme was shown by various methods. Complete dissociation of the apoenzyme is caused by low pH, Na+, or EDTA, whereas the hydroxocobalamin apoenzyme complex does not dissociate under the same conditions. EDTA markedly inhibits the activity of the enzyme whereas other chelating agents have little or no inhibitory effect. Partial reversal of the inhibitory effect of EDTA is achieved by increased concentrations of glycerol or by adding an excess of either subunit, A or B. The experimental evidence suggests that the effect of EDTA on the apoenzyme does not involve a divalent cation.