The mechanism of in sutu reactivation of glycerol-inactivated coenzyme B12-dependent enzymes, glycerol dehydratase and diol dehydratase.

In the previous paper (S. Honda, T. Toraya, and S. Fukui, J. Bacteriol., 143, 1458-1465 (1980)), we reported that the glycerol-inactivated holoenzymes of adenosylcobalamin-dependent glycerol dehydratase and diol dehydratase are rapidly and continually reactivated in toluene-treated cells (in situ) by adenosine 5'-triphosphate (ATP) and divalent metal ions in the presence of free adenosylcobalamin. To elucidate the mechanism of this in situ reactivation, the nature of the binding of various irreversible cobalamin inhibitors to the dehydratases in situ was investigated. In the presence of ATP and Mn2+, enzyme-bound hydroxocobalamin, cyanocobalamin and methylcobalamin were rapidly displaced by added adenosylcobalamin. Without ATP and Mn2+, such displacement did not take place. In contrast, enzyme-bound adeninylbutylcobalamin and adenosylethylcobalamin were essentially not displaceable by the free coenzyme even in the presence of ATP and Mn2+. Inosylcobalamin was a very weak inhibitor irrespective of the presence of ATP and Mn2+. These results indicate that the relative affinity of the enzymes in situ for the cobalamins with simple Co beta ligands was markedly lowered in the presence of ATP and Mn2+, whereas that for the cobalamins with adenine-containing ligands was not. When the glycerol-inactivated holoenzymes in situ were dialyzed against a buffer containing ATP and Mg2+, the inactivated coenzyme moiety dissociated from the enzymes leaving apoproteins. Kinetic evidence was also obtained with the dehydratases in situ that continual displacement of the inactivated coenzyme moiety by adenosylcobalamin takes place during the glycerol dehydration reaction in the presence of ATP and Mn2+. Since the adenosyl group of the bound coenzyme is irreversibly removed from the cobalamin moiety during inactivation by glycerol, all of these data constitute clear evidence that the inactivated holo-dehydratases are reactivated in situ in the presence of ATP and Mn2+ by displacement of the modified coenzyme moiety by free intact adenosylcobalamin (i.e. selective B12-exchange mechanism).