Abstract
When highly purified cobamide-dependent ribonucleotide reductase from Lactobacillus leichmannii is incubated with synthetically prepared 5, 6-dimethylbenzimidazolylcobamide 5'-deoxyadenosyl coenzyme containing tritium attached to carbon atom 5' of the deoxyadenosyl moiety (DBCC-5'-3H), tritium is transferred from DBCC-5'-3H to H2O in a reaction requiring substrate, enzyme, and dithiol reductant. At low enzyme concentrations, the amount of tritium transferred to H2O is stoichiometrically equivalent to the amount of ribonucleotide reduced; hence, substrate-dependent release of tritium from DBCC-5'-3H is a sensitive assay for cobamide-dependent ribonucleotide reductase. When enzyme is incubated with unlabeled cobamide coenzyme in H2O-3H, tritium is transferred to coenzyme as well as to the 2'-deoxyribosyl carbon of the reaction product. The amount of tritium transferred from H2O-3H to product decreases with increasing concentration of unlabeled coenzyme. Coenzyme tritiated in the reductase reaction labels propionaldehyde in the dioldehydrase reaction. The results indicate that cobamide coenzyme functions as an essential hydrogen-transferring agent in the cobamide-dependent ribonucleotide reductase reaction, and that transferred hydrogen attaches to carbon atom 5' of the coenzyme deoxyadenosyl moiety. We conclude that although hydrogen is transferred intermolecularly in the reductase reaction and intramolecularly in the dioldehydrase reaction, the mechanism of coenzyme function is the same in both reactions.
Highlights
The results indicate that cobamide coenzyme functions as an essential hydrogen-transferring agent in the cobamidedependent ribonucleotide reductase reaction, and that transferred hydrogen attaches to carbon atom 5’ of the coenzyme deoxyadenosylmoiety
Assays of radioactivity in the water indicated that tritium is transferred from DBCC-5’-3H to HZ0 in a reaction requiring substrate, enzyme, and dithiol reductant
When enzyme concentration is low enough, cobamide coenzyme functions stoichiometrically, and the amount of tritium released is an approximately linear function of enzyme concentration. These results show that measurement of the substrate-dependent release of tritium from DBCC-5’-3H can serve as a sensitive quantitative assay for ribonucleotide reductase
Summary
The ribonucleotide reductase preparation used was the hydroxylapatite fraction purified from Lactobacillusleiehmannii (ATCC 7830) [4]. Specific activity was 0.6 prnole of CTP reduced per min per mg under standard conditions. The enzyme is monodisperse on sedimentation analysis and essentially homogeneous under conditions of sedimentation equilibrium [5]. Routine assay procedures are described elsewhere [4]. The dioldehydrase preparation used was the E-8 fraction purified from Aerobacteruerogenes (ATCC 8724) [6]. Specific activity was 48 pmoles of propionaldehyde produced per min per mg. Perlman; purified thioreddxin [7] and Mechanism of Action of Cobamide Coenzyme
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