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Use of a mixture of unlabeled and tetradeuterio-,Bmethylaspartate with coenzyme B_(12) dependent β-methylaspartate-glutamate mutase has shown that the hydrogen that migrates becomes one of three equivalent hydrogens during the isomerization. Kinetic isotope effects suggest that cleavage of the bond in the substrate from carbon to that hydrogen which migrates is an important component of the rate-determining step. The evidence also supports the existence of an intermediate which can partition with similar probabilities to β-methylaspartate or to glutamate. Mechanistic implications of these findings are discussed.

The cobalt-carbon bond in alkylcobaloximes, alkylcobalamins, and alkylcobalt derivatives of related chelates is reductively cleaved by thiols and dithiols. The cleavage occurs optimally in neutral or mildly acidic anaerobic medium and involves trans -attack on the cobalt atom by the thiol as the first step. The initial products of the cleavage reaction are carbanionic species which react with protons of the solvent to form alkanes. Consistent with this mechanism, the dealkylation is inhibited by bases competing with thiols for the cobalt coordination site. The methyl carbanionic species generated by the reductive cleavage of methylcobinamide and methylcobaloximes may be trapped by carbon dioxide to form acetate. The latter reaction represents a plausible model for the terminal step of acetate biosynthesis from methylcobalamin and carbon dioxide by cell-free extracts of Clostridium thermoacelicum . Reductive Co C bond cleavage reactions also provide pertinent information concerning the rate-determining step in corrin or coenzyme B 12 -dependent reductase reactions, such as methane biosynthesis by Methanobacillus omelianskii , ribonucleotide reduction by ribonucleotide reductase of Lactobacillus leichmanniii , methylarsine and methylselenide formation by cell extracts of Methanobacterium strain M.O.H. in the presence of arsenite or selenite.

The three-dimensional molecular structure of coenzyme B 12 (5'-deoxyadenosylcobalamin) has been determined by X-ray diffraction. The crystals, as grown from an acetone-water solution and photographed wet, are orthorhombic (space group P 2 1 2 1 2 1 ) with a = 27·93, b = 21·73 and c = 15·34 Å. Four coenzyme molecules (C 72 H 100 O 17 N 18 PCo) and about 68 water molecules make up the unit cell. 3068 Bragg reflexions, extending to a spacing of 0·9 Å, were measured with the crystals in contact with their mother liquor. The intensities were estimated visually from Weissenberg films taken with Cu Kα radiation. The cobalt atoms were easily located from the Patterson synthesis. The structure was solved in three steps, using first cobalt alone, then cobalt and 53 light atoms, and in the third approximation, 106 atoms, which included nearly the full asymmetric unit, except for water and hydrogen. Refinement of the atomic coordinates was accomplished initially by calculation of difference syntheses and finally by differential synthesis. The atomic positions have standard deviations of about 0·04 Å. The conformation of the molecule is very similar to cyanocobalamin. The principal differences are in the orientation of the acetamide and propionamide side chains. Factors which influence the conformation of the corrin nucleus are analysed by comparing several corrinoids of known structure. Features of the molecule which have been examined in detail include the pucker in the pyrroline rings, the bend in the corrin macrocycle, the conformation of the nucleotide and nucleoside moieties and the orientation of the deoxyadenosine moiety with respect to the corrin nucleus. The packing of the molecules and the hydrogen bonding is discussed and compared with that found in the wet and dry vitamin B 12 crystals. Each coenzyme molecule participates in 18 direct intermolecular hydrogen bonds.

We wish to report evidence that the covalent bond in coenzyme B_(12) between the cobalt atom and the methylene group (C-5′) of the nucleoside moiety is cleaved during enzymatic conversion of 1,2-propanediol to propionaldehyde. Acquisition of a third hydrogen satisfies the valence at C-5′ created by the cobalt-C-5′ bond cleavage; C-5′ is thus converted to a methyl group. The experimental result to support this assertion is the observation that mixtures of 1,2-propanediol, propanediol dehydrase and deoxyinosylcobalamin liberate 5′-deoxyinosine to solution.

Summary Cell-free extracts of Rhodopseudomonas spheroides and Rhodospirillum rubrum grown photosynthetically on Co 2+ -supplemented media converted glutamate to β-methylaspartate and further deaminated the latter to form mesaconate. The conversion was inhibited by hog intrinsic factor and the inhibition was restored by coenzyme B 12 . The catalytic activity of cell-free extracts from the cells grown on Co 2+ -deficient media was extremely low and enhanced markedly by the addition of coenzyme B 12 to the reaction system.

The retention of cyanocobalamin, hydroxo-cobalamin, and coenzyme B 12 3 and 28 days after parenteral administration was measured by whole-body monitoring. After 3 days the retention of coenzyme B 12 and hydroxocobalamin was similar, and exceeded that of cyanocobalamin. After 28 days the mean amount of hydroxocobalamin retained was greater than that of cyanocobalamin or coenzyme B 12 . The difference was related to the pattern of loss between 3 and 28 days. The patterns for cyanocobalamin and hydroxocobalamin were similar, and differed from that for coenzyme B 12 . Calculations based on the results suggest that adequate maintenance treatment of uncomplicated vitamin-B 12 deficiency states can be achieved by parenteral administration of 1000 μg. hydroxocobalamin every 4 months or the same dose of cyanocobalamin every 2 months. In the presence of renal or hepatic disease the same dose of cyanocobalamin should be given every 11/2 months and hydroxocobalamin every 2 months.

This chapter describes a method in which the enzyme propanediol dehydratase, obtained from Aerobacter aerogenes , in the presence of coenzyme B 12 catalyzes the oxidation of propanediol to propionaldehyde. The enzyme is also active in the presence of other B 12 coenzymes such as adenyl-cobamide coenzyme and benzimidazolyl coenzyme. The V max with all these coenzymes is the same; therefore, the method described in this chapter should be applicable with these coenzymes, as well. Some extracts of biological material can contain unidentified forms of the coenzyme that may be less active. The application of the method is in the field of biochemistry. With low concentrations of the coenzyme, the rate of propionaldehyde formation is directly proportional to the coenzyme concentration. The amount of propionaldehyde formed per unit time is measured in a colorimetric assay with 2,4-dinitrophenylhydrazine. Vitamin B 12 and related derivatives inhibit the determination of the enzyme. To decrease this inhibition, a 2- to 4-fold excess of enzyme is used.

Publisher Summary This chapter discusses the procedure for the enzymatic preparation of coenzyme B 12 and some of its analogs. Aquo forms of corrinoids are easily converted into their 5'-deoxyadenosyl derivatives under the action of ATP and an appropionate reducing system containing FAD or FMN, and in the presence of an enzyme, ATP: 5'-deoxyadenosyl corrin transferase. Acetone powder of Propionibacterium shermanii is a good source of enzymes and cofactors of this reaction and can be used directly for the preparation of coenzyme B 12 and its analogs. The addition of glucose and mercaptoethanol increases the rate and extent of the conversion. The method is suitable for the preparation of milligram quantities of coenzyme B 12 and its analogs as well as of 5'-deoxyadenosylcobamide and its phosphoric acid derivatives. Reaction products are isolated by phenol extraction and purified by paper electrophoresis and chromatography.

A dead-end ternary complex was formed between propanediol dehydratase ( dl -1,2-propanediol hydro-lyase, EC 4.2.1.28) apoenzyme, coenzyme B 12 , and a substrate analog when a substrate analog such as 1,2-butanediol or styrene glycol was incubated with the apoenzyme and coenzyme B 12 in the presence of potassium ions. The 1,2-diols used did not show any substrate activity, and behaved as weak competitive inhibitors with respect to the substrate. When the true substrate, 1,2-propanediol, was added in excessive amounts to the ternary complex system, the initially bound substrate analog was readily displaced by the substrate and the propanediol dehydratase reaction normally took place. The analog ternary complex was relatively stable to oxygen compared with the holoenzyme, and was much more thermostable than the apoenzyme. Like the holoenzyme and reacting holoenzyme, the ternary complex was photostable under the conditions where free coenzyme B 12 was rapidly photolyzed. The apoenzyme was completely inactivated by incubation with p- chloromercuribenzoate , iodoacetamide, or N- ethylmaleimide , but not with arsenite, suggesting that sulfhydryl groups, but not vicinal ones, are involved at the active site of the enzyme. Treatment with mercaptoethanol or dithiothreitol reversed the inhibition by p- chloromercuribenzoate . In contrast to the mercurial-insensitivity of both the holoenzyme and reacting holoenzyme, the analog ternary complex was considerably sensitive to p- chloromercuribenzoate . Upon Sephadex G-25 gel filtration, dissociation of coenzyme B 12 from the ternary complex occurred more readily than that from the holoenzyme. These results suggest that the apoenzyme-coenzyme-substrate analog ternary complex is a suitable, stable model for the so-called Michaelis complex (intermediate enzyme-substrate complex), and that its structure may be somewhat distorted compared to the holoenzyme and reacting holoenzyme.

The ionophoretic-bioautographic method of detecting cobinamide and cobamide vitamins in millimicrogram quantities has been modified and applied to the detection of cobamide coenzymes in microbial extracts. For the detection of cobamide coenzymes, the cells must be extracted with a neutral buffer in the dark. When cells are extracted with either an alkaline or acid cyanide solution or when the extract is exposed to light, only the cobamide vitamins and other coenzyme decomposition products are observed. Cobamides with the ionophoretic mobility of the cobamide coenzymes have been shown to be present in extracts of Escherichia coli B, E. coli 113-3, Bacillus megatherium, Streptomyces fradiae, Propionibacterium arabinosum, P. shermanii , and Clostridium tetanomorphum . The coenzymes present in several of these organisms have been specifically identified. E. coli 113-3 was shown to convert vitamin B 12 to the 5,6-dimethylbenzimidazolylcobamide coenzyme (coenzyme B 12 ). When grown with cobinamide and benzimidazole, the benzimidazolylcobamide coenzyme was formed.

Nuclear-free liver homogenates were employed to study the rate of incorporation of specifically labeled propionate into methyl-malonate and tricarboxylic acid cycle intermediates. The liver tissue was homogenized in a hypotonic solution. The medium was supplemented with essential coenzymes and incubated for 15min at 37C. Reaction products were isolated by column chromatography. Liver samples were obtained by biopsy from dairy cows representing various stages of lactation and levels of milk production. Considerable variation was found among cows in their ability to metabolize propionate. The incorporation of 14 C-label from propionate into metabolic intermediates beyond methylmalonate was positively correlated with liver vitamin B 12 concentration. This assay criterion was a reflection of methylmalonyl-CoA mutase activity; however, to what extent variations in propionyl-CoA synthetase and carboxylase and racemase enzyme activities affected the magnitude of this parameter could not be assessed. The absence of stimulation by in vitro addition of coenzyme B 12 was interpreted to suggest that the apoenzyme was saturated with coenzyme. A portion of the variation in ability to metabolize propionate could be accounted for by differences in certain lactation characteristics. In general, as the degree of stress of lactation increased, the ability to metabolize propionate decreased.

Publisher Summary This chapter discusses the coenzyme B 12 -dependent mutases causing carbon chain rearrangements. Three B 12 coenzyme-dependent enzymes catalyzing carbon skeleton rearrangements are known: glutamate mutase, methylmalonyl-CoA mutase, and methyleneglutarate mutase. The reactions catalyzed by these enzymes are similar in that they all involve rearrangements in which a substituent group is moved between the α and β positions of a propionate residue, while a hydrogen atom is moved in the opposite direction. The glutamate mutase reaction was discovered during the studies of the path of glutamate fermentation by Clostridium tetanomorphum . The extracts of this bacterium were found to convert glutamate reversibly to ammonia and mesaconate (2-methylfumaric acid)—a carbon, branched chain compound. In the synthesis of propionate by propionibacteria, the mutase reaction functions in the reverse direction from succinyl-CoA to ( R )-methyl-malonyl CoA. A methylmalonyl-CoA racemase is also required in propionate synthesis to form ( S )-methylmalonyl-CoA, which then reacts with pyruvate in a transcarboxylation reaction to give propionyl-CoA and oxalacetate.

Die enzymatische Synthese von Cobamid-Coenzymen ist aus Extrakten, die von Mikroorganismen gewonnen wurden, gut bekannt, nicht aber von Saugetiergeweben. Unsere Befunde mit Cobamid-Coenzymen in der Leber von keimfreien Mausen scheint anzuzeigen, dass die Verwandlung von Vitamin B12 in Coenzymform von Enzymen der Saugetiergewebe katalysiert werden kann.