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

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Abstract
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 dehy-dratase 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 adeninyl-butylcobalamin and adenosylethylcobalamin were essentially not displace-able 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 Coβ 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 vation (10, 21). This inactivation is believed to result from the irreversible cleavage of the activated carbon-cobalt bond of the coenzyme by reaction with oxygen. Figure 4 shows that the oxygen-inactivated glycerol dehydratase was also re-activated in situ in the presence of ATP, Mn2+ and adenosylcobalamin, although the rate of reactivation was rather slow. This slow rate may be attributed at least in part to damage to the apoprotein itself, to extraordinarily tight binding of the compound derived from the coenzyme, or to denaturation of the reactivating system during the oxygen-inactivation procedure. It is known that diol dehydratase is gradually inactivated during dehydration of l, 2-ethanediol (1, 3). Diol dehydratase also undergoes gradual inactivation during catalysis when arabino-adenosylcobalamin is used as a coenzyme instead of adenosylcobalamin (17). The inactivated enzymes thus obtained were also re-activated in situ by ATP, Mn2+ and adenosylcobalamin, although the data are not shown here.

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