Cobamide Coenzyme Research Articles

Electron transfer reactions catalyzed by vitamin B 12 and related compounds: The reduction of dyes and of riboflavin by thiols

The reduction of electron acceptors, such as methylene blue (MB) or ribofiavin, by thiols and other electron donors is catalyzed by traces of vitamin B 12 derivatives, e.g., vitamin B 12a, cyanocobalamin, and Factor B. The catalytic reaction with thiols as reducing agents is of zero order with respect to MB. Rate determining is the reduction of the cobalamin from the Co(III) to the Co(II) state by the mercaptide ion. Various cobalamin model compounds, e.g., cobalt phthalocyanine or cobalt porphyrins, are also active. Agents which compete with the mercaptide ion for coordination sites on cobalt act as inhibitors. Alkylcobalamins and 5′-deoxyadenosyl(dimethylben-zimidazolyl)cobamide coenzyme show little catalytic activity around pH 4–5 but become active at higher pH due to the cleavage of the Co-C bond by mercaptide ion. Since Co-alkylation occurs in vitamin B 12 solutions containing thiols at pH 6–7, strong inhibiting effects are observed on addition of n-alkyl halides to the thiolcobalamin reaction solutions. The extreme sensitivity of the catalytic effects observed suggests that cobalamin derivatives may catalyze in vivo electron transfer reactions at the low physiological concentration level.

Identification and Cobamide Coenzyme-dependent Formation of 3,5-Diaminohexanoic Acid, an Intermediate in Lysine Fermentation

Abstract When 14C-l-lysine is incubated with cell-free extracts of Clostridium SB4, a new basic amino acid is formed. This compound does not accumulate when lysine is fermented in the presence of intrinsic factor. The basic amino acid has been isolated in radiochemically pure form. It is not oxidized by chloramine-T or periodate, it yields about 1 mole of acetic acid per mole in the Kuhn-Roth C-terminal methyl group determination, and it reversibly forms a lactam. The data support the conclusion that this new amino acid, which requires a cobamide coenzyme for formation, is 3,5-diaminohexanoic acid. The basic amino acid is readily fermented to volatile acids by extracts of Clostridium SB4. It appears to be an intermediate in the fermentation of lysine.

Ethanolamine Deaminase, a Cobamide Coenzyme-dependent Enzyme: I. PURIFICATION, ASSAY, AND PROPERTIES OF THE ENZYME

Abstract Ethanolamine deaminase, a clostridial enzyme that catalyzes the cobamide coenzyme-dependent conversion of ethanolamine to ammonia and acetaldehyde, has been obtained as a homogeneous protein. Isolation of the relatively stable enzyme was facilitated by its unique lack of solubility in solutions with an ionic strength greater than 0.11. The specific activities of apparently homogeneous preparations isolated from different batches of cells are slightly different. A spectrophotometric assay for the detection of less than 1 µg of purified enzyme was developed by coupling the formation of acetaldehyde in the deaminase reaction with DPNH oxidation in the presence of added alcohol dehydrogenase. The deaminase reaction has an absolute requirement for cobamide coenzyme. The apparent Km values for α-(5,6-dimethylbenzimidazolyl)cobamide coenzyme, α-benzimidazolyl)cobamide coenzyme, and α-(adenylyl)cobamide coenzyme are 1.5 x 10-6, 1.9 x 10-7, and 7.7 x 10-6 m, respectively. Of several compounds tested, only ethanolamine is a substrate for the enzyme. Potassium ion is required for catalytic activity, whereas Li+ and Na+ are competitive inhibitors. The pH optimum for the reaction is 6.8 to 8.2. p-Chloromercuriphenylsulfonate inhibited the deaminase 46% at 3 x 10-4 m. Like the dioldehydrase described by Lee and Abeles (15), ethanolamine deaminase is inactivated by incubation with cobamide coenzyme in the absence of substrate, and both the deaminase and dioldehydrase reactions have a detectable lag period at low levels of cobamide coenzyme.

Biosynthesis of isoleucine and valine in Mycobacterium tuberculosis H 37 R v

The enzymes involved in the biosynthesis of isoleucine and valine have been shown to be present in cell-free extracts of Mycobacterium tuberculosis H 37R v. In addition to the known enzymes of the pathway, cell-free extracts of this organism contain a new enzyme. When cell-free extracts were incubated with acetolactate and l-ascorbic acid, without reduced nicotinamide adenine dinucleotide phosphate, the isomer of acetolactate, viz., α-keto-β-hydroxyisovalerate, was found to accumulate and was identified by different methods. The reaction is enzymic, and l-ascorbic acid cannot be replaced by other reducing agents such as hydroquinone, 2,6-dichlorophenol indophenol, or glutathione; by derivatives of l-ascorbic acid such as dehydroascorbic acid or dimethyl ascorbic acid; or by cobamide coenzyme. Since the extracts also isomerize α-acetohydroxybutyrate to α-keto-β-hydroxy-β-methylvalerate, the enzyme catalyzing the reaction has been termed “acetohydroxy acid isomerase.” This is the first time that the presence of acetohydroxy acid isomerase has been reported in any biological system and that a specific metabolic role has been assigned for l-ascorbic acid. The extract also possesses reductase activity to convert α-keto-β-hydroxyisovalerate to α,β-dihydroxyisovalerate in the presence of reduced nicotinamide adenine dinucleotide phosphate.

Ethanolamine Deaminase, a Cobamide Coenzyme-dependent Enzyme: II. PHYSICAL AND CHEMICAL PROPERTIES AND INTERACTION WITH COBAMIDES AND ETHANOLAMINE

Abstract A purified preparation of clostridial ethanolamine deaminase was shown to be homogeneous by sedimentation in the ultracentrifuge and by disc gel electrophoresis. The enzyme has an s20,w of 14.5 S and a weight average molecular weight of 520,000 g. Treatment with 5 m guanidine hydrochloride results in dissociation of subunits with a weight average molecular weight of 51,000 g. Amino acid analysis of native and performic acid-oxidized deaminase indicated the presence of 3 moles of half-cystine residues per minimum molecular weight of 28,700 g. Titration with 5,5'-dithiobis(2-nitrobenzoic acid) yielded only 1 mole of free sulfhydryl group per mole of native enzyme (520,000 g) but in the presence of sodium lauryl sulfate 15 sulfhydryl groups per molecule were titratable. The spectrum of the enzyme contains a prominent peak at 470 mµ, with other peaks at 357, 410, and 530 mµ. The chromophore was resolved from the enzyme by treatment with acid-ammonium sulfate. On resolution, the 470 mµ peak of the enzyme disappeared and the dissociated chromophore had a spectrum similar to that of hydroxocobalamin and related cobamides. The chromophore was identified as an α-(adenylyl)cobamide by paper chromatography and paper electrophoresis. Between 1.35 and 3.1 moles of cobamide were found per mole of native enzyme. On resolution, the specific activity of the deaminase, when tested in the presence of added cobamide coenzyme, increased an average of 38%. Hydroxocobalamin, cyanocobalamin, and methylcobalamin were shown to be irreversible inhibitors of ethanolamine deaminase. Incubation of resolved enzyme with varying amounts of hydroxocobalamin resulted in complete inhibition of the enzyme after the binding of 2 to 3 moles of cobamide per mole of enzyme. The enzyme was able to bind about 7 moles of hydroxocobalamin per mole. In both the presence and absence of ethanolamine, incubation of the enzyme resulted in the slow appearance of a spectral peak at 352 mµ. Oxidation and Schmidt degradation of acetaldehyde produced by the enzymatic deamination of 1-14C-ethanolamine hydrochloride showed that the aldehyde carbon of the acetaldehyde is derived from the carbinol carbon of ethanolamine.

Anaerobic degradation of lysine: V. Some properties of the cobamide coenzyme-dependent β-lysine mutase of Clostridium sticklandii

β-Lysine mutase, a cobamide coenzyme-dependent enzyme system from Clostridium sticklandii that converts β-lysine (3,6-diaminohexanoate) to 3,5-diaminohexanoate, has been fractionated into two protein components. One of these, an acidic, orange protein, contains a tightly bound cobamide, and the other, less acidic, appears to be a sulfhydryl protein. The partially purified enzyme system requires as cofactors a cobamide coenzyme, ATP, a mercaptan, FAD, pyruvate, Mg 2+, and a monovalent cation such as K + or Rb +. The reaction is reversible.

Studies on the Mechanism of Hydrogen Transfer in the Cobamide Coenzyme-dependent Dioldehydrase Reaction

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.

A cobamide coenzyme dependent migration of the ϵ-amino group of D-lysine

Application of the amino acid analyzer is described for the separation, identification, and estimation of several uncommon dibasic amino acids that are eluted with a 0.35 m, pH 5.28, sodium citrate buffer between lysine and ammonia, namely, 2,5-diaminohexanoate, threo- and erythro-3,5-diaminohexanoate, 3,6-diaminohexanoate (β-lysine) and 2,4-diaminopentanoic acid. Conditions are also given for estimating d- and l-β-lysine as their l-glutamyl peptides, and for estimating the enantiomers of threo- and erythro-3,5-diaminohexanoate as the l-glutamyl derivative of the corresponding lactams.

The Mechanism of Action of Cobamide Coenzyme in the Ribonucleotide Reductase Reaction

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.

Methylmalonic Acidemia a new inborn error of metabolism which may cause fatal acidosis in the neonatal period

A new inborn error of metabolism characterized by severe metabolic acidosis, polyuria, dehydration, emaciation and the urinary excretion of large amounts of methylmalonic acid is described. Two siblings of the patient have died in the neonatal period with clinical symptoms strongly resembling those of our patient. Our studies indicate that the metabolic block is located in the degradation of methylmalonic acid to succinic acid.Valine was shown to be a precursor of methylmalonic acid in accordance with the known metabolic formation of this acid. Small amounts of valine were metabolized to CO2, suggesting that the metabolic defect is incomplete. The blood concentration, body burden, distribution volume, serum half life and renal clearance of methylmalonic acid were determined.Treatment with cyanocobalamin and with cobamide coenzyme did not appear to influence the course of the disease.A diet low in isoleucine, valine, threonine and methionine significantly reduced the urinary excretion of methylmalonic acid...

Transfer of hydrogen from cobamide coenzyme to water during enzymatic ribonucleotide reduction

A purified preparation of combamide-dependent ribonucleotide reductase from Lactobacillus leichmannii catalyzes an exchange of hydrogen between water and cobamide coenzyme in the course of CTP reduction. The exchange is dependent upon the reductase reaction.

Methylmalonic acid excretion in vitamin B12 defciency.

methylmalonic acid was first isolated from pooled human urine in 1957 by Thomas and Stalder and convincing evidence has since been obtained to show that in man the isomerization of l‐methylmalonyl‐Co A to succinyl‐Co A is dependent on the B12 coenzyme dimethylbenzimidazolyl cobamide (White, 1962). Indeed, Cannata, Focesi, Mazumder, Warner and Ochoa (1965) showed recently that l‐methylmalonyl‐Co A mutase bears two molecules of cobamide coenzyme firmly attached to its molecule. In a series of 30 patients Cox and White (1962) detected excessive methylmalonic acid in the urine of all patients with serum vitamin B12 levels below 140 pg./ml., their lower limit of normal. Patients with normal serum B12 levels excreted up to 4 mg. of methylmalonic acid in 24 hours. Their series included six patients who were treated with intramuscular hydroxocobalamin (1000 μg.). In five of these, excretion fell to normal within 3 days, and on the fifth day after injection in the sixth patient. Barness, Young, Mellman, Khan and Williams (1963) reported one vitamin B12 deficient patient in whom the urinary excretion of methylmalonic acid reached 500 mg. in 24 hours. Recently Khan, Williams, Barness, Young, Shafer, Vivacqua and Beaupre (1965) have described seven out of nine patients with pernicious anaemia in whom methylmalonic acid excretion persisted for some months after the institution of B12 therapy. This occurred despite full clinical and haematological remissions and in four patients, normal serum vitamin B12 levels.The present study was undertaken to determine the value of urinary methylmalonic acid excretion as a screening test for vitamin B12 deficiency. The estimation of methylmalonic acid was carried out by a rapid thin layer chromatographic technique, suitable for routine laboratory use. Normal controls and patients suffering from general haematological and medical disorders were screened as well as patients suffering from megaloblastic anaemia. In several patients the excretion of methylmalonic acid was followed for a short period after B12 therapy.

Enzymatic Conversion of Vitamin B12s to a Cobamide Coenzyme, α-(5,6-Dimethylbenzimidazolyl)deoxyadenosylcobamide (Adenosyl-B12)

Abstract 1. Clostridium tetanomorphum contains an adenosylating enzyme, that converts B12s (a reduced form of vitamin B12 in which the cobalt atom is in the +1 oxidation state) to the cobamide coenzyme, adenosyl-B12 (B12s + adenosine triphosphate → adenosyl-B12 + tripolyphosphate). 2. The enzyme has been purified approximately 300-fold, with an over-all recovery of about 40% by treatment of sonically disrupted cells with protamine, followed by selective heat denaturation and chromatography on diethylaminoethyl cellulose. The purified enzyme is free from B12a reductase and glutamate mutase. 3. The pH optimum for the enzyme is 8.0. Michaelis constants for B12s and ATP are 1.0 and 1.6 x 10-5 m, respectively. B12s cannot be replaced by B12r or B12a (forms of B12 in which the oxidation state of cobalt is +2 and +3, respectively). ATP can be replaced by cytidine triphosphate and, to a lesser degree, by the triphosphates of uridine, guanosine, and inosine. Mn++ is required as divalent cation; Co++ and Mg++ are less effective. 4. With the purified enzyme the forward reaction can be demonstrated spectrophotometrically by means of the increase in absorbance at 525 mµ as B12s is converted to adenosyl-B12. No evidence could be obtained for reversal of the reaction.

Comparison of reactivity between d-propanediol and l-propanediol in intramolecular oxidation-reduction catalyzed by dioldehydrase requiring cobamide coenzyme

The substrate stereospecificity of a dioldehydrase requiring DBC-coenzyme was investigated with d(−)- and l(+)-propanediols as the substrates. Both isomers could be converted to propionaldehyde, but the d-isomer was 1.5–2 times more reactive than the l-form. An inhibition was observed if a high concentration of the l-form or the dl-form was used; moreover, the reactivity of the d-form was depressed by the simultaneous addition of the l-form.

The Absolute Configuration of Methylmalonyl Coenzyme A and Stereochemistry of the Methylmalonyl Coenzyme A Mutase Reaction

Abstract Propionyl coenzyme A was carboxylated with epimerase-free carboxylase, and the resulting methylmalonyl coenzyme A (a) was reduced with Raney nickel. Isolation of (-)-(R)-β-hydroxyisobutyric acid N-phenylcarbamate (11) from the reaction mixture showed that methylmalonyl coenzyme A (a) has the (S) configuration. Propionyl coenzyme A was also converted to deuteriosuccinate with a D2O extract of a beef liver mitochondrial acetone powder. The deuteriosuccinate had a plain positive optical rotatory dispersion curve corresponding to the (S) configuration. It was concluded that (S)-methylmalonyl coenzyme A, formed by carboxylation of propionyl coenzyme A, was epimerized in D2O to (RS)-methylmalonyl(coenzyme A)-2-D, and that (R)-methylmalonyl(coenzyme A)-2-D was isomerized to succinyl coenzyme A with net retention of configuration of C-2. Since, in contrast, the glutamate mutase reaction proceeds with inversion, the lack of stereochemical uniformity in these two otherwise closely related rearrangements suggests that they may involve at least two steps, one or more of which are analogous, but at least one taking place with different stereochemistry. It is suggested that the cobamide coenzyme releases an electrophilic 5'-deoxyadenosyl group (perhaps reversibly to a methionyl residue on the enzyme), and that the resulting Co:i complex participates in the rearrangement by removal and transfer of a proton or other cationic fragment.

The Clostridial Fermentations of Choline and Ethanolamine: II. REQUIREMENT FOR A COBAMIDE COENZYME BY AN ETHANOLAMINE DEAMINASE

The Clostridial Fermentations of Choline and Ethanolamine: II. REQUIREMENT FOR A COBAMIDE COENZYME BY AN ETHANOLAMINE DEAMINASE

Requirement of ribonucleotide reductase for cobamide coenzyme, a product of ribosomal activity.

Requirement of ribonucleotide reductase for cobamide coenzyme, a product of ribosomal activity.

CONVERSION OF CYANO- AND HYDROXO-COBALAMIN IN VIVO INTO CO-ENZYME FORM OF VITAMIN B12 IN THE RAT.

SINCE 5,6-dimethylbenzimidazolyl cobamide co-enzyme (DBCC) was shown to be one of the active forms of vitamin B12 by Barker et al. in 1959 (ref. 1), its biochemical co-enzymatic activity (conversion of glutamate to methyl aspartate, methyl malonyl–CoA to succinyl–CoA, and 1,2-diols to aldehydes) and its physiological metabolism (tissue distribution, excretion and absorption) have been investigated2–8. It is now believed that vitamin B12 exists as a co-enzyme form in the liver, and takes part in the transformation of methyl malonyl–CoA to succinyl–CoA (ref. 9). On the other hand, enzymatic synthesis of DBCC from B12 derivatives has been confirmed by several workers at the bacterial enzymatic level. It has been reported that the liver and kidney homogenate of rat could convert cyanocobalamin (CN–B12) to co-enzyme B12 in vitro10. But the only report indicating that CN–B12 or hydroxocobalamin (OH–B12) can be converted to co-enzyme form in vivo, on the quantitative base, is Fenrych's short communication reporting the conversion of CN–B12 into the co-enzyme form in rabbit11. Our preliminary report concerning this conversion, which was obtained by 57Co-labelling of the DBCC fraction in rat liver following the intravenous administration of 57Co-labelled CN–B12 and 57Co-labelled OH–B12 in rat, will be described here.

Effect of light-irradiation and adenosine on the activity of a dioldehydratase-requiring cobamide coenzyme

Effect of light-irradiation and adenosine on the activity of a dioldehydratase-requiring cobamide coenzyme

A rapid and simplified method for preparing purified intrinsic factor

A rapid and simplified method for preparing large quantities of purified hog intrinsic factor by a batchwise process on DEAE-cellulose is described. This procedure results in highly concentrated material with essentially 100% yield of intrinsic factor activity. The purified material is active at 0.5 mg or less (100 × National Formulary), binds 5.0 μg of vitamin B 12 per milligram, and is suitable for studies of cobamide coenzyme dependent reactions and as starting material for the isolation and chemical characterization of pure intrinsic factor.