Abstract
Abstract When dl-1,2-propanediol-1-3H is converted to propionaldehyde in the presence of dioldehydrase and cobamide coenzyme, tritium is transferred to the coenzyme. The tritiated coenzyme so obtained transfers tritium to the reaction product when reacted with dl-1,2-propanediol and apoenzyme. The coenzyme is tritiated exclusively at the C-5' position of the adenosyl moiety. The location of tritium was established by chemical degradation and further confirmed by showing that chemically synthesized cobamide coenzyme, containing tritium at the C-5' position, transferred tritium to the product when added to enzyme and unlabeled substrate. The conversion of dl-1,2-propanediol-1-3H to propionaldehyde proceeds with inter- and intramolecular tritium transfer. Approximately 1% of the tritiated substrate reacts by intramolecular transfer, i.e. the hydrogen abstracted from C-1 of a substrate molecule is found in the α position of the aldehyde derived from that molecule. A partial reaction occurs, as evidenced by tritium exchange between tritiated coenzyme and propionaldehyde or acetaldehyde under conditions in which no net reaction occurs. The results obtained have led to the following tentative reaction sequence. Hydrogen is abstracted from C-1 of dl-1,2-propanediol and transferred to the coenzyme, where it becomes equivalent with at least one, but probably both, hydrogens of the C-5' position. This results in the formation of a reduced form of the coenzyme and a molecule derived through the oxidation of the substrate. In a subsequent step the hydrated form of propionaldehyde is formed by a transfer of hydrogen from the reduced coenzyme to the intermediate derived from the substrate.
Highlights
The results obtained have led to the following tentative reaction sequence
Enzymatic Transfer of Tritium from dl-1,2-Propanediol-1 -3H to DBC Coenzyme-When dl-1,2-propanediol-l-3H is converted to propionaldehyde in the presence of dioldehydrase and DBC coenzyme, tritium is incorporated into the coenzyme
The detailed procedure used for the transfer of tritium from substrate to the coenzyme, dissociation of the enzyme-coenzyme complex, and reisolation of radioactive coenzyme, is described under
Summary
Coenzyme, and Substrates-Dioldehydrase was isolated from Awobacter aerogenes as previously described [1]. In which substrate-free enzyme tias required, it was necessary to remove d&1,2-propanediol which is present in the purified enzyme as a stabilizing agent. In these experiments dioldehydrase was dialyzed for 2 hours against 0.01 M. KzHPOl at O-5”, treated with 1 pg of DBC coenzymei per 200 units of enzyme at 37” for 10 min, and dialyzed an additional 2 hours against 0.01 M K2HP04 in the cold. Crystalline DRC coenzyme was prepared by the procedure of Pailes and Hogenkamp [10]. Commercial dl-1,2-propanediol, ethylene glycol, propionaldehyde, and acetaldehyde were redistilled before use.
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