Ligand exchange reactions of diol dehydrase-bound cobalamins and the effect of the nucleoside binding.

The inactive complex of diol dehydrase with hydroxocobalamin was resolved by treatment with SO2-3, followed by dialysis to remove SO2-3, giving the apoenzyme which was reconstitutable into catalytically active holoenzyme upon addition of adenosylcobalamin ("re-activation"). Spectral evidence showed that the enzyme-bound hydroxocobalamin undergoes a Co beta-ligand exchange reaction forming sulfitocobalamin. Sulfitocobalamin was bound to diol dehydrase only loosely, and therefore dissociated from the enzyme. In contrast, neither the enzyme-hydroxocobalamin-5'-deoxyadenosine nor the enzyme-hydroxocobalamin-adenosine complex was resolved and thus re-activated by this procedure. It was shown spectroscopically that the hydroxocobalamin in these complexes does not react with SO2-3, or even with CN-, indicating that the OH group in the Co beta-position was blocked spatially by these enzyme-bound nucleosides. Neither O2-inactivated holoenzyme nor the holoenzyme inactivated suicidally by glycerol or 1,2-ethanediol during dehydration reaction was also re-activated by the same procedure. The complex of the enzyme with cyanocobalamin or methylcobalamin was not resolvable by the SO2-3 treatment. This was because these cobalamins bound to the enzyme were not subject to a ligand exchange reaction with SO2-3.