Immobilized diol dehydrase and its use in studies of cobalamin binding and subunit interaction.
Coenzyme B12 dependent diol dehydrase from Aerobacter aerogenes was immobilized by covalent binding to CNBr-activated Sepharose 4B. The Sepharose-bound enzyme exhibited a markedly high catalytic activity, viz., 75-95% of the specific activity of the original free enzyme. The apoenzyme acquired much greater stability to heat by immobilization. No significant difference between the immobilized and free enzymes was observed in the following properties: the affinity for coenzyme B12; the sensitivity to a sulfhydryl-modifying agent; the absolute requirement for a certain monovalent cation, such as K+, for catalysis; the susceptibility toward oxygen upon incubation with coenzyme B12 in the absence of substrate. These results suggest that the structure and function of the enzyme are not significantly influenced by immobilization on Sepharose. The immobilized enzyme was found to provide a convenient method for a study of ligand interaction with the enzyme. The subunit interaction between two dissimilar subunits, components F and S, was investigated using the component S immobilized on CNBr-activited Sepharose and free component F, and it was demonstrated that the substrate (1,2-propanedoil) promotes the hybrid formation between component F and component S, but K+ alone rather retarded the subunit association to some extent. Na+ markedly weakens the forces which bind the subunits together. The relationship between cobalamin binding and subunit structure is also discussed.
D001051:Apoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D003038:Cobamides / Q000378:metabolism*; D004355:Drug Stability; D004754:Enterobacter / Q000201:enzymology*; D004755:Enterobacteriaceae / Q000201:enzymology*; D006358:Hot Temperature; D006836:Hydro-Lyases / Q000378:metabolism*; D046911:Macromolecular Substances; D010084:Oxidation-Reduction; D011188:Potassium / Q000494:pharmacology; D011408:Propanediol Dehydratase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011485:Protein Binding; D012685:Sepharose; D012964:Sodium / Q000494:pharmacology
D001051:Apoenzymes; D003038:Cobamides; D046911:Macromolecular Substances; D012685:Sepharose; D012964:Sodium; D006836:Hydro-Lyases; D011408:Propanediol Dehydratase; D011188:Potassium