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Metabolic Role of Vitamin B12
Metabolic Role of Vitamin B,, HERBERT WEISSBACH
AND
ROBERT T. TAYLOR
Laboratory of Clinical Biochemistry, National Heart Institute, National Institutes of Health, Bethesda, Maryland
I. Introduction . . . . . . . . . . . . . . 11. Cleavage and Synthesis of the Carbon-to-Cobalt Bond . . . . 111. Enzyme-Catalyzed Reactions Requiring 5'-Deoxyadenosyl Cobamides IV. Mechanism of the 5'-Deoxyadenosyl-h-Dependent Reactions . . . . . . . . . . . . V. Methyltransferase Reactions VI. Role of Vitamin EL in Animal Metabolism . . . . . . . . . . . . . . . . . . . . . References
. 395 . 396
.
.
. . .
398 401 403 408 409
I. INTRODUCTION Two decades have passed since the isolation of the antipernicious anemia factor, vitamin B,, (Rickes e t al., 1948; Smith, 1948). Although the vitamin after its isolation was implicated in a variety of biochemical reactions ranging from a role in one-carbon metabolism to the synthesis of macromolecules, i t was not until 1958 that a coenzymatic role of the vitamin was clearly established. At that time Barker and co-workers (1958) demonstrated that a derivative of pseudovitamin B,, was involved in the conversion of glutamic acid to P-methylaspartat'e catalyzed by cell-free extracts of Clostridium tetanomorlJhum. A similar derivative, containing 5,6-dimethylbenzimidazole, was isolated from C . tetanomorphum, animal liver, and propionic acid bacteria (Weissbach e t al., 1959). The exact structure of the BIZ-coenzyme shown in Fig. 1 was subsequently elucidated by Lenhert and Hodgkin (1961) by X-ray crystallography. These elegant studies demonstrated the presence of a 5'-deoxyadenosyl group in the axial ligand occupied by the cyanide anion in the vitamin. The nucleoside is linked to the cobalt via the 5'-carbon atom of its deoxyribosyl moiety, and it is now well documented that both the chemical and the biological reactivity of the B,,-coenzyme (or other alkyl cobamides) reside in this carbon-to-cobalt bond. Chemical studies on the cleavage and the synthesis of the carbon-to-cobalt bond have yielded valuable data on the nature of this linkage and have provided a means to investigate the role of the alkyl cobamides in enzymatic reactions. 395
396
HERBERT WEISSBACH A S D ROBERT T. TAYLOR
11. CLEAVAGE AND SYNTHESIS OF
THE
CARBON-TO-COBALT BOND
The light sensitivity of alkyl cobamides was first noted during the initial isolation of the B,,-coenzyme (Barker et al., 1960; Weissbach et al., 1960). Brady and Barker (1961) subsequently showed that light degradation of the coenzyme under anaerobic conditions resulted in a homolytic cleavage of the carbon-to-cobalt bond to yield BlZr, a reduced form of the vitamin-containing divalent cobalt (Co++)(Hogenkamp et al., 1963; Dolphin et al., 1964). B,,,, although stable in the absence of oxygen, readily oxidizes to hydroxy-B,, in air (Eq. 1).
\I
/I
R
/
C03+
\
Alkyl-cobamide (R = alkyl group)
light d
\I
/I
/
coz+
\
B*2=
0 2 L
\I
/I
OH
/
co3+
\
Hydroxy-Biz
Photolytic decomposition of the carbon-to-cobalt bond is a rather specific characteristic of all the alkyl cobamides that have been examined
397
METABOLIC ROLE OF VITAMIN BIZ
and has been used to detect enzyme-bound alkyl cobamides in studies on the cobamide-dependent methyltransferase reactions. The chemical synthesis of various alkyl cobamides was reported by both Smith et al. (1962) and Bernhauer et al. (1962). It involves a twoelectron reduction of the trivalent cobalt in the vitamin to a Co’ derivative referred to as B,,,. Because of its monovalent cobalt atom, B,,, exhibits strong nucleophilic properties and readily reacts with chemical alkylating agents to form the corresponding alkyl cobamides. A chemical synthesis of the simple alkyl cobamide, methyl-B,,, is summarized in Eq. ( 2 ) . \I
OH
co
/I\
-
/’
2e
Hydroxy-BIZ
\I
/’
Cof
/I\
CH*I d
dark
Bizs
\I
CHI
/’
co
(2)
/I\
Methyl-BIz
The enzymatic synthesis of a carbon-to-cobalt bond is quite analogous to the chemical synthesis shown above. For example, the synthesis of BIZ-coenzyme which is catalyzed by extracts of Propionibacterium shermanii (Brady et al., 1962) and C . tetanomorphum (Weissbach et al., 1961 ; Peterkofsky and Weissbach, 1963) was observed to require both a reducing system and ATP, the latter supplying the 5’-deoxyadenosyl group. Using the C. tetanomorphum (Peterkofsky and Weissbach, 1963) enzyme system, it was found that the 3-phosphates of ATP are released as inorganic tripolyphosphate (Eq. 3) ~
\I
/I
OH
/
Co
\
5’-Deo adenosyl
+ATP
Hydroxy-Blz
system
\ 7c o/
/I
\
5’-Deoxyadenosyl-B1, (B12-Coenzyme)
+
P-P-P
(3)
Tripolyphosphate
The enzyme system obtained from propionic acid bacteria is very similar except that inorganic pyrophosphate and orthophosphate were the observed coproducts (Brady et al., 1962). In the overall enzymatic reaction, as in the chemical synthesis of the carbon-to-cobalt bond, the reducing system is needed to convert the cobamide substrate t o B,,,, which then accepts a 5’-deoxyadenosyl group from ATP. Although reduced flavin (Brady et al., 1962) and reduced ferredoxin (Weissbach et al., 1966) can presumably supply the reducing power for B,,, formation, a small molecular weight thioprotein has been reported to be the natural reducing agent in C. tetanomorphum (Walker et al., 1967). Once B,,, has been formed, a separate “adenosylating enzyme” (Vitols et al., 1966) catalyzes the deoxyadenosyl group transfer;
398
HERBERT WEISSBACH AND ROBERT T. TAYLOR
Mn” is required for this transfer reaction (Vitols et al., 1966). It should be stressed that in both the chemical and the enzymatic synthesis of the carbon-to-cobalt bond a reducing system (to reduce the cobamide) and an alkyl group donor are required.
111. ENZYMECATALYZED REACTION s REQUIRING 5’-DEOXYADENOSYL COBAMIDES
The enzymatic reactions requiring vitamin B,, derivatives can be divided into two major groups: (a) those that utilize 5’-deoxyadenosylB,, and involve a hydrogen transfer, and (b) those that involve a methyl group transfer, probably via a methyl-B,, enzyme. The reactions requiring 5’-deoxyadenosyl-BI2 are shown in Fig. 2 and are briefly described below. 1. Glutamate Mutase (Isomerase)
Studies on this reaction by Barker and colleagues led to the isolation of the first coenzyme form of vitamin B,, (Barker et al., 1958). The reaction is freely reversible and involves a transfer of a hydrogen from the C-4 position of L-glutamate to the C-3 position, and a migration of the glycine residue to the C-4 position to form threo-@-methyl-Laspartate. No free intermediates have been detected (Barker et al., 1964) and there is no incorporation of deuterium from the solvent into the products (Iodice and Barker, 1963). Two distinct protein components, E (Suzuki and Barker, 1966) and S (Switzer and Barker, 1967), have been purified and both are essential for glutamate mutase activity. 2. Methylmalonyl-CoA Mutase
This reaction is very similar to the glutamate mutase reaction in that both a hydrogen transfer and a cleavage of a carbon-to-carbon bond with an alkyl group migration occur. A hydrogen moves from the C-3 position of succinyl CoA to the C-2 position while the CoA thioester carboxyl group migrates to the C-3 position (H. G. Wood et al., 1964; Eggerer et al., 1960). In this reaction, also, free intermediates have not been detected and there is no incorporation of hydrogen from the solvent into the products.
3. Diol Dehydrase Reaction Abeles and Lee (1961) first described this reaction in Aerobacter aerogenes. The enzyme catalyzes an irreversible conversion of either ethylene glycol or propylene glycol (propane-l,2-diol) to acetaldehyde or propionaldehyde, respectively. Hydrogen migrates from the C-1 position to the C-2 position and there is no exchange with hydrogen from the
METABOLIC ROLE O F VITAMIN Biz
iiq
399
1 . Glutamate mutase
LI~~--C-COOH
2.
FH.H HC -C-COOH I
-
/
NHz
COOH
Methylmalonyl- CoA m u t a s e
0 H II HC-C-SCOA COOH
COOH
3. Glycal dehydrase H
H HCH
I
HC=O 4.
Ribonucleotide r e d u c t a s e
LoJ
Base-C-C-C-C-C-0-P-P-P
-
H
lea
H Base-C-C-C-C-C-0-P-P-P H I H H OH
&HhHH
5. Ethanolamine deaminase H HC-NH,
=
I
HC-OH H 6. 8-Lysine
-
H
HTH HC=O
f
NH,
3,5-Diaminohexanoic acid
0 H H H H H l l HC-CC-C-C-C-C-OH I H H NH, I H NH2
-
0 H H H H H l l HC-C-C-C-C-C-OH H I H l H NH, NH,
FIG.2. BIa-Coensyme-dependentreactions.
solvent (Brownstein and Abeles, 1961). It is of interest to note that although an alkyl migration does not occur in this reaction (unlike the mutase reactions), Retey e t al. (1966) have shown that the oxygen atom on the C-2 position of propane-1,2-diol moves to the C-1 position of the propionaldehyde that is formed. Both a hemiacetal form of lactaldehyde
400
HERBERT WEISSIUCH AND ROBERT T. TAYLOR
and the hydrated form of the aldehyde product have been postulated by these authors to be intermediates in the conversion of propane-l,2-diol to propionaldehyde (Eq. 4 ) . H
-+
I HC-OH I
COB^
HCOH
I
CH3
--t
HC-OH
H
1;. I
OH
I
CHO
HC-OH -HCH
H-CoB12 CHI
I
-HzO
-CH2
AH3
I
(4)
CH,
Smiley and Sobolov (1962) have reported a reaction similar to the glycol dehydrase reaction using glycerol as a substrate. 4. Ribonucleotide Reductase Reaction
This reaction was first described by Blakley and Barker in extracts of Lactobacillus leichmunii (Blakley and Barker, 1964) and i t explains a t the enzymatic level the known ability of deoxyriboncleosides to alleviate the vitamin B,, growth requirement of this organism (Kitay et al., 1949). L. leichmanii ribonucleotide reductase requires a Blz-coenzyme and utilizes only nucleoside triphosphates (Abrams, 1965; Blakley et al., 1965; Blakley, 1966; Beck et ul., 196613) as opposed to the ribonucleotide reductase in Escherichia coli, which does not require a cobamide and is specific for nucleoside diphosphates (Reichard, 1962). A L. Zeichnzanii thioprotein reducing system (Orr and Vitols, 1966) similar to the thioredoxin system in E. coli (Moore and Reichard, 1963) normally functions in the cobamide-dependent reaction, although reduced lipoic acid alone can serve as a source of hydrogen in this system. Therefore, this reaction, unlike all the other BIZ-coenzyme-dependent reactions in Fig. 2, involves a net reduction with the incorporation of hydrogen from the solvent into the product. Studies on the mechanism of the hydrogen incorporation (Gottesman and Beck, 1966; Blakley et al., 1966) have shown that the solvent hydrogen is incorporated into the C-2’ position of the product, with retention of configuration (Batterham et al., 1967), suggesting that the hydroxyl group in the C-2’ position is displaced by a hydride ion via a stereospecific SN, mechanism. It should be noted that B,,-coenzyme-dependent enzymes show no apparent uniformity with regard to the stereochemistry of the reactions that they catalyze. Thus, in the glutamate mutase (Sprecher et al., 1966b) and glycol dehydrase (Zagalak et al., 1966) reactions inversion of substrate configuration occurs; whereas, in the methylmalonyl-CoA mutase (Sprecher et al., 1966a) and ribonucleotide reductase (Batterham e t al., 1967) reactions the configuration of the substrate is retained.
METABOLIC ROLE O F VITAMIN Biz
401
5. Ethanolamine Dehydrase Reaction This reaction was first reported by Bradbeer (1965) and has been studied recently by Babior (1967). Although detailed studies on the mechanism of this reaction have not been reported, it may be analogous to the glycol dehydrase reaction except that a deamination instead of a dehydration occurs. Again there is no incorporation of deuterium from the solvent into acetaldehyde (Babior, 1967). 6. Conversion of p-Lysine to 3,5-Diaminohexanoic Acid Stadtman showed that two strains of clostridia were able to ferment lysine to acetate, butyrate, and ammonia (Stadtman, 1963). Indirect experiments suggested a role of a cobamide coenzyme in this pathway. Studies by Costilow et al. (1966) identified 3,6-diaminohexanoate (Plysine) as the first product of lysine fermentation; it is formed in a pyridoxal phosphate-dependent reaction. The subsequent conversion of P-lysine to 3,5-diaminohexanoic acid requires 5'-deoxyadenosyl-B12 (Stadtman and Renz, 1967; Bray and Stadtman, 1968) and can be viewed as a migration of the amino group on C-6 to C-5 with a simultaneous transfer of hydrogen from C-5 to (2-6.
Iv. MECHANISMO F
REACTIONS It is now apparent that all the reactions shown in Fig. 2 involve a TH E 5'-DEOXYADENOSYL-B12-DEPENDENT
transfer of hydrogen. With the exception of the ribonucleotide reductase reaction, the hydrogen is transferred to an adjacent carbon with a migration in the reverse direction of a group (oxygen, alkyl, etc.) to the carbon which has lost its hydrogen. In the ribonucleotide reductase system (described in detail elsewhere in this volume) a hydrogen is transferred from the reducing agent into the product. The carrier of the hydrogen in the glycol dehydrase reaction has received the most attention and the role of deoxyadenosyl-B,, in this process is now well established. Abeles and Zagalak (1966) first demonstrated that hydrogen transfer in the glycol dehydrase reaction is not a direct intramolecular transfer despite the failure to incorporate hydrogen from the solvent into the products. In these studies, a radioactive substrate (l-3H-propane-1,2-diol) and a nonradioactive substrate (ethylene glycol) were simultaneously decomposed by the enzyme and radioactive acetaldehyde (3Hon C-2) was formed along with the expected radioactive product, propionaldehyde (3H on C-2). The ability to form a radioactive product from a nonradioactive substrate in these experiments indicated that the radioactive hydrogen from propane-l,2-diol must have been transferred to a position on the enzyme-coenzyme compIex which con-
402
HERBERT WEISSBACH AND ROBERT T. TAYLOR
tained a t least 2 equivalent hydrogens. Removal of a nonradioactive hydrogen from the enzyme-coenzyme complex would leave 3H on the enzyme-coenzyme complex which could subsequently be transferred to a molecule of unlabeled ethylene glycol yielding 3H-acetaldehyde. Abeles and Frey (1966) and Frey et al. (1967a) showed that the coenzyme was acting as a hydrogen carrier in the reaction by isolating radioactive coenzyme after incubating the enzyme with l-3H-propane-1,2-dio1. The isolated radioactive coenzyme could transfer its tritium to product in a subsequent reaction using an unlabeled substrate. I n other confirmatory experiments, chemically synthesized coenzyme, labeled with 3Hin the C-5’ position, transferred all its tritium t o propionaldehyde in the enzymatic oxidoreduction of unlabeled propane-l,2-diol (Frey and Aheles, 1966; Frey et al., 1967a). I n the glycol dehydrase reaction only about 1% of the l-3H-propane-1,2-dio1 is converted to propionaldehyde by the abstraction of a hydrogen from the C-1 position followed by its return to the C-2 position of the same molecule (Frey et al., 1967a). The essential role of the hydrogen a t the C-5’ position of the coenzyme has been further demonstrated by the studies of Retey and Arigoni (1966). Csing coenzyme labeled with 3H on the C-5’ position by means of the diol dehydrase reaction, they were able to demonstrate the transfer of tritium to products during catalysis of the methylmalonyl-CoA mutase reaction. The ribonucleotide reductase reaction is atypical, however, compared to the other reactions shown in Fig. 2 in that tritium on the C-5’ of chemically synthesized BIZ-coenzyme does not become incorporated into the deoxyribonucleotide product. Instead, tritium was transferred quantitatively from the (3-5’ position into water in a reaction system that contained ribonucleotide enzyme, substrate, and a reducing agent (Beck et al., 1966a). It has been suggested (Beck e t al., 1966a), therefore, that the enzyme catalyzes a reversible hydrogen transfer between the reducing agent (reduced lipoate) and the coenzyme-enzyme complex so that rapid equilibration of the hydrogen on the reducing agent with water could explain the results that were obtained. This was substantiated when Hogenkamp et al. (1967) observed the exchange of tritium between water and the C-5’ position even when the substrate ribonucleotides were replaced by any one of the several deoxyribonucleotides, especially deoxyGTP. Although the incubation components needed for solvent hydrogencoenzyme exchange closely mimic those needed for the overall reduction (Fig. 2) (Beck et al., 1966a; Hogenkamp et aE., 1967; Abeles and Beck, 1967) and the rate of hydrogen exchange is comparable to the overall rate of reduction (Hogenkamp et al., 1967), it is still not definitely established that the coenzyme actually functions as an intermediate hydrogen carrier in this system. It is particularly important to note t ha t in both the glycol dehydrase
METABOLIC ROLE O F VITAMIN Biz
403
and the ribonucleotide reductase systems both hydrogens on the (2-5’ position of the B,,-coenzyme-enzyme complex arc labilized (Frey et al., 1967b) during catalysis. Yet, these two (3-5’ hydrogens are not sterically equivalent on the unbound, free B,,-coenzyme-containing tritium in the C-5’ position. This was shown conclusively when both isomers of BIZcoenzyme-C-5’ (3H) were prepared and each transferred all its tritium to propionaldehyde in the glycol dehydrase system. When the ribonucleotide reductase reaction was carried out in pure deuterium oxide, 1.6 atoms of deuterium per molecule were incorporated a t the C-5’ position of the coenzyme. These findings establish that a t some point in B,,-coenzymedependent reactions the two C-5’ hydrogens became sterically equivalent, perhaps by a cleavage of the carbon-cobalt bond atom a t the C-5’ position. I n this connection Tamao et al. (1967) have attributed the reduced activity of 10-C1-BIZ-coenzyme (ie., chloro on the C-10 of the corrin nucleus) in the glycol dehydrase system to a polarization of the carboncobalt bond, the electrons being more attracted to the cobalt. They have consequently suggested that a carbon-cobalt bond cleavage occurs to give a 5’-deoxyadenosyl carbanion ( eCH, - R) . It must be concluded that in all the B,,-coenzyme-dependent reactions, hydrogen from either the substrate or a reducing agent (as in the case of the ribonucleotide reductase reaction) is transferred to the C-5’ position of the coenzyme bound to the enzyme. With the exception of the ribonucleotide reductase reaction, the hydrogen in the enzyme-coenzyme complex is subsequently transferred to the product without equilibrating with hydrogen of the solvent.
V. METHYLTRANSFERASE REACTIONS Three cobamide-dependent reactions are now known which do not require 5‘-deoxyadenosyl-B,,. Instead, a tightly bound cobamide prosthetic group is functional, and the enzymes involved catalyze methyl group transfer reactions (Fig. 3) in which methyl-BIZ probably serves as a protein-bound intermediate. Included in this group of reactions are the syntheses of methionine, methane, and acetate. I n all cases, N5-methylH,-folate, as well as methyl-B,,, can serve as the direct methyl donor, although the folate derivative is probably the natural substrate.
1. Methionine Synthesis This reaction appears to be the simplest of the cobamide-dependent methyltransferase reactions and has been investigated in the greatest detail. The studies by Woods and co-workers (Guest e t al., 1960, 1964; Kisliuk and Woods, 1960; Foster et nl., 1964) as well as Buchanan and collaborators (Larrabee e t al., 1961; Hatch et al., 1961; Takeyama et al., 1961) contributed largely to the understanding of the requirements and
404
HERBERT WEISSITACH AXD ROBERT T. TAYLOR 1.
Methionine synthesls N5-Methyl-H,-Folate
2.
+
Hornocysteine _c Methionine
+
H,-Folate
Methane synthesis N5-Methyl-H,-Folate -Methane
3. Acetate synthesis NS-Methyl-H,-Folate
+
C0,--Acetate
7
Methionine
CH,
2‘0’
I’
‘
Methyl- B
l
Methane q Acetate
FIG.3. Cobamide-dependent methyltransferme reactions.
the general characteristics of the reaction in Escherichia coli. The reaction, shown below, involves a methyl group transfer from N5-methyl-H,folate (Larrabee et a;., 1961) t.o homocysteine to form methionine and H,-folate (Eq. 5.) N6-Methyl-Ha-folate
AMe Protein > Methionine reducing system
+ Homocysteine
+ H4-Folate
(5)
A cobamide-containing protein (Guest et al., 1960), a reducing system (Guest et al., 1960; Hatch et al., 1961), and catalytic levels of S-adenosylmethionine (AMe) (Mangum and Scrimgeour, 1962; Rosenthal and Buchanan, 1963; Foster et al., 1964; Weissbach et al., 1963) are essential. The observation by Guest et al. (1962) that the enzyme also catalyzed the transfer of a methyl group from methyl-B,, to homocysteine focused attention on the possibility that methyl-B,, might be an enzyme-bound intermediate in methyl group transfer from Nj-methyl-H,-folate t o homocysteine. I n the reaction sequence suggested below, the folate derivative methylates a reduced cobamide species on the enzyme to form a methyl-B,,-enzyme and H,-folate. Then a subsequent transfer of the methyl group to homocysteine occurs to yield methionine and to regenerate a reduced B,,-enzyme (Eq. 6 ) . H4-Folate
N6-Methyl-H4-folate
+\I
/I
/
Co+
\
Enzyme t
AMe
+
CHI
\ I /’ Co /I \
Enzyme
Komocysteine
I
> Methionine
(6)
METABOLIC ROLE O F VITAMIN Biz
405
In this two-step reaction sequence, AMe is pictured as being required only for alkylation of the enzyme by N5-methyl-H,-folate. Extensively purified preparations of the enzyme have been obtained from extracts of E. coli (Takeyama and Buchanan, 1961; Taylor and Weissbach, 1967a). The B,, enzyme in such preparations is salmoncolored and displays an absorption spectrum (475 mp maximum) that closely resembles the spectrum of B,,, (Taylor and Weissbach, 1967a) or a B1,-thiol complex (Dolphin and Johnson, 1963). These spectral similarities and the fact that sulfito-B,, is obtained (Takeyama and Buchanan, 1961; Ertel e t al., 1968) upon alcohol extraction of the B,,-protein suggest that the cobamide may be linked to an SH group on the enzyme. Studies utilizing millimicromole amounts of enzyme and either radioactive N5-methyl-14C-H,-folate or methyl-I4C-AMe have shown that both compounds react with the bound cobalamin to yield a methyl-Blzenzyme (Taylor and Weissbach, 196713, 1968a). A reducing system is required for both methyl donors to alkylate the bound cobamide, and in addition, AMe is needed for methyl group transfer from N5-methyl-H4folate to the enzyme-bound cobamide. Of interest was the finding that a methyl-14C-B,, enzyme was stable to light (as measured by the release of volatile I4C or radioactive formaldehyde) until the protein was acidified to below pH 2.5 (Taylor and Weissbach, 1968a). Definitive proof that a methyl-B12-enzyme was formed in these experiments was obtained by ethanol extraction of the bound cobamide followed by identification of the radioactive chromophore as methyl-"C-B,,. If the methyl-B1,enzyme is isolated by procedures that do not cause denaturation (e.g., Sephadex gel filtration), it is active catalytically, and it can transfer its methyl-I4C group to homocysteine to form methyl-*4C-methionine (Taylor and Weissbach, 1 9 6 7 ~ )This . reaction proceeds to completion in air without the requirement of either a reducing system or exogenous AMe. Essentially, these foregoing observations have been made recently by Stavrianopoulos and Jaenicke (1967)) who also used highly purified E. coli transmethylase. Recent studies on the time course of methylation of the bound cobamide in the presence of both AMe and N5-methyl-H,-folate have shown that AMe rapidly methylates the enzyme-bound cobamide first and methyl groups from the folate donor subsequently replace the methyl groups on the cobamide which originated from AMe (Taylor and Weissbach, 196813). I n this regard, it has also been possible t o show that an enzyme methylated with AMe, and then reisolated, will catalyze limited methyl group transfer from N5-methyl-H,-folate to homocysteine in the absence of exogenous AMe and a reducing system (Taylor and Weissbach, 196%). This "priming" action occurs only under conditions in which
406
HERBERT WEISSHACH AND ROBERT T. TAYLOR
AMe is able to methylate the cobamide on the enzyme and has also been accomplished with methyl iodide. These observations suggest that AMe may function in its normal manner as a methyl donor but methylate only the initial molecules of BIZenzyme. The bound cobamide attached initially to the enzyme is present in an inactive form, perhaps linked to a sulfur on the protein. AMe or an efficient chemical methylating agent such as methyl iodide (Taylor and Weissbach, 1967d) might react with the enzyme-bound cobamide to form a methyl-B,,-enzyme; hence, the first methyl group on the enzyme would arise from AMe. Transfer of this methyl group to homocysteine as a carboiiium ion would yield a reduced cobamide species (Cot) on the enzyme which could rapidly accept subsequent methyl groups only from N5-methyl-H,-folate. N5-Methyl-H,-folate would then continue to supply methyl groups for methionine synthesis until the cobamide on the enzyme was oxidized back to its original inactive form. Activation, or priming, would thus be accomplished by AMe in a catalytic manner. 2. Methane Formation
A cobamide derivative was implicated in methane formation after the initial observation by Blaylock and Stadtman that methyl-B,, was converted to methane by extracts of Methanosarcina barkeri (Blaylock and Stadtman, 1963). Additional studies by Blaylock and Stadtman (1964), as well as by Wolin et al. (1963) using extracts of M e t h a n ~ b a ~ i l ~ u s omeliansfcii, have further suggested the participation of a protein-bound cobamide in methane formation. Direct evidence for the involvement of a cobamide containing enzyme has come from the studies of J. M. Wood and Wolfe (1966a), who isolated from M . omelianskii a methane-forming enzyme containing 5-hydroxybenzimidazolyl cobamide (factor 111). In addition t o a reducing system, ATP is required for methane formation from methyl-B,, (Wolin et al., 1963), N5-methyl-H,-folate (J. M. Wood et al., 1965), and methyl-B,, analogs (J. M. Wood et al., 1966). J. M. Wood and Wolfe (1966b) reported that 1 mole of ATP was decomposed per mole of methane formed. Blaylock and Stadtman (1966) have shown that methanol, which is a substrate for methane formation, can donate, in the presence of ATP and a reducing system, a methyl group to B,,, forming methyl-B,,. While the available evidence suggests that a free methyl cobamide is not an intermediate in methane formation, an enzyme-bound methyl cobamide (as pictured in the methionine reaction) is probably involved. The enzymes that catalyze methionine and methane formation can both be inhibited by reacting the protein-bound chromophore with propyl iodide to form a propyl-cobamide enzyme (Brot and Weissbach, 1965; J. M. Wood and Wolfe, 1966a; Taylor and Weissbach,
407
METABOLIC ROLE O F VITAMIN Biz
1967e). Restoration of catalytic activity to a propylated enzyme by exposure to visible light strongly suggests that the cobalt atom is directly involved in these enzymatic processes and that a common reaction mechanism exists. It is noteworthy, however, that cobalt methyl derivatives have not been found in extracts of methane-forming organisms (Lezius and Barker, 1965). 3. Acetate Synthesis
Barker and Kamen (1945) first demonstrated that Clostridium thermoaceticum utilized COz for the total synthesis of acetate. Poston et d. (1964) provided evidence that this reaction involved a cobamide derivative. The authors observed that the formation of the methyl group of acetate from CO, in cell-free extracts was inhibited by intrinsic factor and that methyl-B,, could serve as a methyl donor for the methyl group of acetate. The exact sequence by which CO, is incorporated into acetate and the role of cobamide derivatives in this process is still unknown. Irion and Ljungdahl (1965) have isolated a wide variety of corrinoid compounds from C . thermoaceticum including methyl and deoxyadenosyl derivatives of cobyric acid and 5-methoxybenzimidazolyl cobamide (factor IIIm). Exposure of the cells to 14C0, resulted in the rapid labeling of cobalt methyl groups in methylcobyric acid, methyl factor IIIm, and traces of carboxymethyl corrinoid (Ljungdahl et aZ., 1965). These and additional isotopic experiments (Ljungdahl et al., 1967) indicate that a protein-bound cobalt methy1J4C corrinoid is formed upon incubation with 14C0, and that this methyl-14C corrinoid is then converted to a proteinbound, cobalt carboxymethyl derivative which can be reductively cleaved to acetate. Two protein fractions and ferredoxin have been separated from extracts of C . thermoaceticum (Poston and Stadtman, 1967) and are required in combination with CoA and pyruvate to convert methyl-I4Ccobalamin to radioactive acetate. A pathway summarizing these experiments has been postulated by Ljungdahl et al. (1966) and is shown in Eq. (7).
I
1
auction
CHICOOH
Thus, in all the methyl transferase reactions, a methyl-B1, protein is currently considered to be an intermediate.
408
HERBERT WEISSE'ACH
AND ROBERT T. TAYLOR
VI. ROLEOF VITAMINB,
IN
ANIMAL METABOLISM
Studies with pernicious anemia patients have suggested that vitamin B,, is required primarily for DNA synthesis which is essential for normal development of the mature red blood cell in the bone marrow. Considering the reactions that were described in the preceding sections, attention is immediately focused on the participation of cobamide derivatives in ribonucleotide reduction and the formation of methionine, two possible enzymatic sites of BIZaction that would affect DNA synthesis. Although only limited data are available (Moore and Hurlbert, 1962), there is, as yet, no evidence in support of the view that ribonucleotide reduction in animal systems requires a cobamide derivative. I n fact, of the known reactions that require 5'-deoxyadenosyl-B,, (Fig. 2) , only the methylmalonyl-CoA mutase reaction has been demonstrated in animal tissues (Lengyel et al., 1960). Of the methyl transferase reactions shown in Fig. 3, there is substantial evidence that the synthesis of methionine requires a cobamide derivative in animal tissues (Loughlin et al., 1964; Dickerman e t al., 1964). In addition, it is well established that there is a nutritional interrelationship among folic acid, vitamin B,,, and onecarbon metabolism (Bennett et al., 1951; Machlin et al., 1952; Sunde et al., 1951; Fox et al., 1956, 1961; Silverman and Pitney, 1959). A summary of various tetrahydrofolic acid derivatives which depicts their role in one-carbon metabolism is shown in Fig. 4. It is seen that tetrahydrofolate is directly involved in the synthesis of purines, thymidylate, and methionine. I n vitamin B12deficiency, the rate of methionine synthesis might N"'- Formyl-Folate-H,
Histidine -FIGLU Serine Folate-H,
N5,lo-Methylene-Folate-H,
N5-Methyl- Folate-H,
FIG.4. Metabolism of folic acid derivatives.
METABOLIC ROLE O F VITAMIN Biz
409
be diminished and the ability to regenerate H,-folate from N5-methyl-Hafolate would be impaired. If the synthesis of methionine from homocysteine is a major means by which H,-folate is regenerated in the body, a deficiency of vitamin B,, would markedly alter folate metabolism and result in an accumulation of N5-methyl-H,-folate a t the expense of H4folate and its methylene and methenyl derivatives. Direct determination of folate compounds in animals on vitamin B,,-deficient diets have confirmed that the partition of folk acid derivatives is changed in vitamin B,, deficiency (Norohna and Silverman, 1961). I n addition, the increased excretion of formiminoglutamic acid (FIGLU) observed in vitamin BIZ deficiency (Fox e t al., 1961 ; Silverman and Pitney, 1959) is presumably caused by a deficiency of H,-folate. Based on these data, i t is plausible that many of the effects of vitamin B,, deficiency in bone marrow are related to an altered metabolism of €I,-folate. I n contrast, the central nervous system lesion seen in pernicious anemia could be related to the methylmalonyl-CoA mutase reaction. REFERENCES Abeles, R. H., and Beck, W. S. (1967). J . Biol. Chem. 242, 3589. Abeles, R. H., and Frey, P. A. (1966). Federation Proc. 25, 1639. Abeles, R. H., and Lee, H. A. (1961). J . B i d . Chem. 236, 2347. Abeles, R. H., and Zagalak, B. (1966). J . Biol. Chem. 241, 1245. Abrams, R. (1965). J . Biol. Chem. 240, PC3697. Babior, B. (1%7). Federation Proc. 26, 344. Barker, H. A., and Kamen, M. D. (1945). Proc. Natl. Acad. Sci. U S . 31, 219. Barker, H. A., Weissbach, H., and Smyth, R. D. (1958). Proc. Natl. Acad. Sci. U.S. 44, 1093.
Barker, H. A., Smyth, R. D., Weissbach, H., Munch-Petersen, A., Toohey, J. I., Ladd, J. N., Volcani, B. E., and Wilson, R. M. (1960). J . Biol. Chem. 235, 480. Barker, H. A,, Suzuki, F., Iodice, A. A., and Rooze, V. (1964). Ann. N.Y. Acad. Sci. 122, 644.
Batterham, T. J., Ghambeer, R. K., Blakley, R. L., and Brownson, C. (1967). Biochemistry 6, 1203. Beck, W. S., Abeles, R. H., and Robinson, W. K. (1966a). Biochem. Biophys. Res. Commun. 25, 421. Beck, W. S., Goulian, M., Larsson, A., and Reichard, P. (196613). J . Biol. Chem. 241, 2177.
Bennett, M. A,, Joralemon, J., and Halpern, P . E. (1951). J . Biol. Chem. 193, 285. Bernhauer, K., Miiller, O., and Miiller, G. (1962). Biochem. 2. 336, 102. Blakley, R. L. (1966). J . Biol. Chem. 241, 176. Blakley, R. L., and Barker, H. A. (1964). Biochem. Biophys. Res. Commun. IS, 391. Blakley, R. L., Ghambeer, R. K., Nixon, P. F., and Vitols, E . (1965). Biochem. Biophvs. Res. Commun. 20, 439. Blakley, R. L., Ghambeer, R. K., Batterham, T. J., and Brownson, C. (1966). Biochem. Biophys. Res. Commun. 24, 418. Blaylock, B. A., and Stadtman, T. C. (1963). Biochem. Biophys. Res. Commun. 11, 34.
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HERBERT WEISSRACACI-I A S D ROBERT T. TAYLOR
Blaylock, B. A,, and Stadtman, T. C. (1964). Ann. N . Y . Acad. Sci. 112, 799. Blaylock, B. A., and Stadtman, T. C. (1966). Arch. Biochem. Biophys. 116, 138. Bradbeer, C. (1965). J. Biol. Chem. 240, 4675. Brady, R. O., and Barker, H. A. (1961). Biochem. Biophys. Res. Commun. 4, 373. Brady, R. O., Castanera, E. G., and Barker, H . A. (1962). J . Biol. Chem. 237, 2325. Bray, R. C., and Stadtman, T. C. (1968). J. Biol. Chem. 243, 381. Brot, N., and Weissbach, H. (1965). J. Biol. Chem. 240, 3064. Brownstein, A. M., and Abeles, R. H. (1961). J. Biol. Chem. 236, 1199. Costilow, R. N., Rochovansky, 0. M,, and Barker, H. A. (1966). J. Biol. Chem. 241, 1573. Dickerman, H., Redfield, B. G., Bieri, J., and Weissbach, H. (1964). J. Biol. Chem. 239, 2545. Dolphin, D. H., and Johnson, A. W. (1963). Proc. Chem. SOC.p. 311. Dolphin, D. H., Johnson, A. W., and Rodrigo, R. (1964). Ann. N.Y. Acad. Sci. 112, 590. Eggerer, H., Stadtman, E. R., Overath, P., and Lynen, F. (1960). Biochem. Z.333, 1. Ertel, R. E., Brot, N., Taylor, R. T., and Weissbach, H. (1968). Arch. Biochem. Biophys. 125, 353. Foster, M. A., Dilworth, M. J., and Woods, D. D. (1964). Nature 201, 39. Fox, M. R., Ortiz, L. O., and Briggs, G. M. (1956). Proc. SOC.Exptl. Biol. M e d . 93, 501. Fox, M. R., Ludwig, W. J., and Baroody, M. C. (1961). Proc. SOC.Expptl. Biol. Med. 107, 723. Frey, P. A., and Abeles, R. H. (1966). J. Biol. Chem. 241, 2732. Frey, P. A,, Essenberg, M. K., and Abeles, R. H. (1967s). J. Biol. Chem. 242, 5369. Frey, P. A., Kerwar, S. S., and Abeles, R. H. (1967b). Biochem. Biophys. Res. Commun. 29, 873. Gottesman, M . M., and Beck, W. S. (1966). Biochem. Biophys. Res. Commun. 24, 353. Guest, J. R., Helleiner, C. W., Cross, M. J., and Woods, D. D. (1960). Biochem. J . 76, 396. Guest, J. R., Friedman, S., Woods, D. D., and Smith, E. L. (1962). Nature 195, 340. Guest, J. R., Friedman, S., Woods, D. D., and Smith, E . L. (1964). Ann. N . Y . Acarl. Sci. 112, 774. Hatch, F. T., Larrabee, A. R., Cathou, R. E., and Buehanan, J. M. (1961). J. Biol. Che.m. 236, 1095. Hogenkamp, H. P. C., Barker, H. A,, and Mason, H. S. (1963). Arch. Biochem. Bwphys. 100, 353. Hogenkamp, H. P. C., Ghambeer, R. K., Brownson, C., and Blakley, R. L. (1967). Biochem. J. 103, 5c. Iodice, A., and Barker, H. A. (1963). J . Biol. Chem. 238, 2094. Irion, E., and Ljungdahl, L. (1965). Biochemistry 4, 2780. Kisliuk, R. L., and Woods, D. D. (1960). Biochem. J. 75, 467. Kitay, E., MacNutt, W. S., and Snell, E . E. (1949). J. Biol. Chem. 177, 993. Larrahee, A. R., Rosenthal, S., Cathou, R. E., and Buchanan, J. M. (1961). J. A m . Chem. SOC.83, 4094. Lengyel, P., Mazumder, R., and Ochoa, S. (1960). Proc. Naptl. Acatl. Sci. U S . 46, 1312. Lenhert, P. G., and Hodgkin, D. C. (1961). Nature 192, 937. Leeius, A. G., and Barker, H. A. (1965). Biochemistry 4, 510.
METABOLIC ROLE O F VITAMIN Biz
411
Ljungdahl, L., Irion, E., and Wood, H. G. (1965). Biochemistry 4, 2771. Ljungdahl, L., Irion, E., and Wood, H. G. (1966). Federation Proc. 25, 1642. Ljungdahl, L., Glatzle, D., Goodyear, J., and Wood, H. G. (1967). Bacterial. Proc. p. 128 (abstr.). Loughlin, R. E., Elford, H. L., and Buchanan, J. M. (1964). J. Biol. Chem. 239, 2888. Machlin, L. J., Denton, C. A., and Bird, H. R. (1952). Poultry Sci. 31, 110. Mangum, J. H., and Scrimgeour, K. G. (1962). Federation Proc. 21, 242. Moore, E. C., and Hurlbert, R. B. (1962). Biochim. Biophys. Acta 55, 651. Moore, E. C., and Reichard, P. (1963). J. Biol. Chem. 238, PC2244. Norohna, J. M., and Silverman, N. (1961). 2nd European Symp. Vit.-BI2 and Intrinsic Factor, 728, Enke, Stuttgart. Orr, M. D., and Vitols, E. (1966). Biochem. Biophys. Res. Commun. 25, 109. Peterkofsky, A., and Weissbach, H. (1963). J. Biol. Chem. 238, 1491. Poston, J. M., Kuratomi, K., and Stadtman, E. R. (1964). Ann. N.Y. Acad. 5%. 112, 804.
Poston, J. M., and Stadtman, E. R. (1967). Biochem. Biophys. Res. Commun. 26, 550.
Reichard, P. (1962). J. Biol. Chern. 237, 3513. Retey, J., and Arigoni, D. (1966). Ezperientiu 22, 783. Retey, J., Umani-Ronchi, A,, Seibl, J., and Arigoni, D. (1966). Ezperientiu 22, 502. Rickes, E. L., Brink, N. G., Konivszy, F. R., Wood, T. R., and Folkers, K. (1948). Science 107, 396. Rosenthal, S., and Buchanan, J. M. (1963). Acta Chem. Scand. 17, Suppl. 1, 288. Silverman, M., and Pitney, A. J. (1959). J. Biol. Chem. 223, 1179. Smiley, K. L., and Sobolov, M. (1962). Arch. Biochem. Biophys. 97, 538. Smith, E. L. (1948). Nature 161, 638. Smith, E. L., Mervyn, L., Johnson, A. W., and Shaw, N. (1962). Nature 194, 1175. Sprecher, M., Clark, M. J., and Sprinson, D. B. (1966a). J . Biol. Chem. 241, 872. Sprecher, M., Smitzer, R. L., and Sprinson, D. B. (1966b). J. Biol. Chem. 241, 864. Stadtman, T. C. (1963). J . Biol. Chem. 238, 2766. Stadtman, T. C., and Renz, P. (1967). Federation Proc. 26, 343. Stavrianopoulos, J., and Jaenicke, L. (1967). European J. Biochem. 3, 95. Sunde, M. L., Waibel, P. E., Cravens, W. W., and Elvehjem, C. A. (1951). Poultry Sci. 30, 668. Suzuki, F., and Barker, H. A. (1966). J. Biol. Chem. 241, 878. Switzer, R. L., and Barker, H. A. (1967). J. Biol. Chem. 242, 2658. Takeyama, S., and Buchanan, J. M. (1961). J. Biochem. ( T o k y o ) 49, 578. Takeyama, S., Hatch, F. T., and Buchanan, J. M. (1961). J . Biol. Chem. 236, 1102. Tamao, Y., Morikawa, Y., Shimizu, S., and Fukui, S. (1967). Biochem. Biophys. Res. Commun. 28, 692. Taylor, R. T., and Weissbach, H. (1967a). J . Biol. Chem. 242, 1502. Taylor, R. T., and Weissbach, H. (1967b). Biochem. Biophys. Res. Commun. 27, 398. Taylor, R. T., and Weissbach, H. (1967~).Arch. Biochem. Biophgls. 119, 572, Taylor, R. T., and Weissbach, H. (1967d). J. Biol. Chem. 242, 1517. Taylor, R. T., and Weissbach, H. (1967e). J . Biol. Chem. 242, 1509. Taylor, R. T., and Weissbach, H. (1968a). Arch. Biochem. Biophys. 123, 109. Taylor, R. T., and Weissbach, H. (1968b). To be published. Vitols, E., Walker, G. A., and Huennekens, F. M. (1966). J. Biol. Chem. 241, 1455. Walker, G. A., Murphv, S., Schmidt, R. R., and Huennekens, F. M. (1967). Federation Proc. 26, 343.
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HERBERT WEISSgACH AiVD ROBERT T. TAYLOR
Weissbach, H., Toohey, J. I., and Barker, H. A. (1959). Proc. Natl. Acad Sci. U.S. 45, 521. Weissbach, H., Ladd, J. H., Volcani, B. E., Smyth, R. D., and Barker, H. A. (1960). J . Biol. Chem. 235, 1462. Weissbach, H., Redfield, B. G., and Peterkofsky, A. (1961). J . Biol. Chem. 236, PC40. Weissbach, H., Peterkofsky, A., Redfield, B. G., and Dickerman, H. (1963). J . Biol. Chem. 238, 3318. Weissbach, H., Brot, N., and Lovenberg, W. (1966). 1. Biol. Chem. 241, 317. Wolin, M. J., Wolin, E. A., and Wolfe, R. S. (1963). Biochem. Biophys. Res. Commun. 12, 464. Wood, H. G., Kellermeyer, R. W., Stjernholm, R., and Allen, S. H. G. (1964). Ann. N . Y . Acad. Sci. 112, 661. Wood, J. M., and Wolfe, R. S. (1966a). Biochemistry 5, 3598. Wood, J. M., and Wolfe, R. S. (1966b). J . Bacteriol. 92, 696. Wood, J. M., Allam, A. M., Brill, W. J., and Wolfe, R. S. (1965). J . Biol. Chem. 240, 4564. Wood, J. M., Wolin, M. J., and Wolfe, R. S. (1966). Biochemistry 5, 2381. Zagalak, B., Frey, P. A., Karabatsos, G. L., and Abeles, R. H. (1966). J. Biol. Chem. 241, 3028.
AND
ROBERT T. TAYLOR
Laboratory of Clinical Biochemistry, National Heart Institute, National Institutes of Health, Bethesda, Maryland
I. Introduction . . . . . . . . . . . . . . 11. Cleavage and Synthesis of the Carbon-to-Cobalt Bond . . . . 111. Enzyme-Catalyzed Reactions Requiring 5'-Deoxyadenosyl Cobamides IV. Mechanism of the 5'-Deoxyadenosyl-h-Dependent Reactions . . . . . . . . . . . . V. Methyltransferase Reactions VI. Role of Vitamin EL in Animal Metabolism . . . . . . . . . . . . . . . . . . . . . References
. 395 . 396
.
.
. . .
398 401 403 408 409
I. INTRODUCTION Two decades have passed since the isolation of the antipernicious anemia factor, vitamin B,, (Rickes e t al., 1948; Smith, 1948). Although the vitamin after its isolation was implicated in a variety of biochemical reactions ranging from a role in one-carbon metabolism to the synthesis of macromolecules, i t was not until 1958 that a coenzymatic role of the vitamin was clearly established. At that time Barker and co-workers (1958) demonstrated that a derivative of pseudovitamin B,, was involved in the conversion of glutamic acid to P-methylaspartat'e catalyzed by cell-free extracts of Clostridium tetanomorlJhum. A similar derivative, containing 5,6-dimethylbenzimidazole, was isolated from C . tetanomorphum, animal liver, and propionic acid bacteria (Weissbach e t al., 1959). The exact structure of the BIZ-coenzyme shown in Fig. 1 was subsequently elucidated by Lenhert and Hodgkin (1961) by X-ray crystallography. These elegant studies demonstrated the presence of a 5'-deoxyadenosyl group in the axial ligand occupied by the cyanide anion in the vitamin. The nucleoside is linked to the cobalt via the 5'-carbon atom of its deoxyribosyl moiety, and it is now well documented that both the chemical and the biological reactivity of the B,,-coenzyme (or other alkyl cobamides) reside in this carbon-to-cobalt bond. Chemical studies on the cleavage and the synthesis of the carbon-to-cobalt bond have yielded valuable data on the nature of this linkage and have provided a means to investigate the role of the alkyl cobamides in enzymatic reactions. 395
396
HERBERT WEISSBACH A S D ROBERT T. TAYLOR
11. CLEAVAGE AND SYNTHESIS OF
THE
CARBON-TO-COBALT BOND
The light sensitivity of alkyl cobamides was first noted during the initial isolation of the B,,-coenzyme (Barker et al., 1960; Weissbach et al., 1960). Brady and Barker (1961) subsequently showed that light degradation of the coenzyme under anaerobic conditions resulted in a homolytic cleavage of the carbon-to-cobalt bond to yield BlZr, a reduced form of the vitamin-containing divalent cobalt (Co++)(Hogenkamp et al., 1963; Dolphin et al., 1964). B,,,, although stable in the absence of oxygen, readily oxidizes to hydroxy-B,, in air (Eq. 1).
\I
/I
R
/
C03+
\
Alkyl-cobamide (R = alkyl group)
light d
\I
/I
/
coz+
\
B*2=
0 2 L
\I
/I
OH
/
co3+
\
Hydroxy-Biz
Photolytic decomposition of the carbon-to-cobalt bond is a rather specific characteristic of all the alkyl cobamides that have been examined
397
METABOLIC ROLE OF VITAMIN BIZ
and has been used to detect enzyme-bound alkyl cobamides in studies on the cobamide-dependent methyltransferase reactions. The chemical synthesis of various alkyl cobamides was reported by both Smith et al. (1962) and Bernhauer et al. (1962). It involves a twoelectron reduction of the trivalent cobalt in the vitamin to a Co’ derivative referred to as B,,,. Because of its monovalent cobalt atom, B,,, exhibits strong nucleophilic properties and readily reacts with chemical alkylating agents to form the corresponding alkyl cobamides. A chemical synthesis of the simple alkyl cobamide, methyl-B,,, is summarized in Eq. ( 2 ) . \I
OH
co
/I\
-
/’
2e
Hydroxy-BIZ
\I
/’
Cof
/I\
CH*I d
dark
Bizs
\I
CHI
/’
co
(2)
/I\
Methyl-BIz
The enzymatic synthesis of a carbon-to-cobalt bond is quite analogous to the chemical synthesis shown above. For example, the synthesis of BIZ-coenzyme which is catalyzed by extracts of Propionibacterium shermanii (Brady et al., 1962) and C . tetanomorphum (Weissbach et al., 1961 ; Peterkofsky and Weissbach, 1963) was observed to require both a reducing system and ATP, the latter supplying the 5’-deoxyadenosyl group. Using the C. tetanomorphum (Peterkofsky and Weissbach, 1963) enzyme system, it was found that the 3-phosphates of ATP are released as inorganic tripolyphosphate (Eq. 3) ~
\I
/I
OH
/
Co
\
5’-Deo adenosyl
+ATP
Hydroxy-Blz
system
\ 7c o/
/I
\
5’-Deoxyadenosyl-B1, (B12-Coenzyme)
+
P-P-P
(3)
Tripolyphosphate
The enzyme system obtained from propionic acid bacteria is very similar except that inorganic pyrophosphate and orthophosphate were the observed coproducts (Brady et al., 1962). In the overall enzymatic reaction, as in the chemical synthesis of the carbon-to-cobalt bond, the reducing system is needed to convert the cobamide substrate t o B,,,, which then accepts a 5’-deoxyadenosyl group from ATP. Although reduced flavin (Brady et al., 1962) and reduced ferredoxin (Weissbach et al., 1966) can presumably supply the reducing power for B,,, formation, a small molecular weight thioprotein has been reported to be the natural reducing agent in C. tetanomorphum (Walker et al., 1967). Once B,,, has been formed, a separate “adenosylating enzyme” (Vitols et al., 1966) catalyzes the deoxyadenosyl group transfer;
398
HERBERT WEISSBACH AND ROBERT T. TAYLOR
Mn” is required for this transfer reaction (Vitols et al., 1966). It should be stressed that in both the chemical and the enzymatic synthesis of the carbon-to-cobalt bond a reducing system (to reduce the cobamide) and an alkyl group donor are required.
111. ENZYMECATALYZED REACTION s REQUIRING 5’-DEOXYADENOSYL COBAMIDES
The enzymatic reactions requiring vitamin B,, derivatives can be divided into two major groups: (a) those that utilize 5’-deoxyadenosylB,, and involve a hydrogen transfer, and (b) those that involve a methyl group transfer, probably via a methyl-B,, enzyme. The reactions requiring 5’-deoxyadenosyl-BI2 are shown in Fig. 2 and are briefly described below. 1. Glutamate Mutase (Isomerase)
Studies on this reaction by Barker and colleagues led to the isolation of the first coenzyme form of vitamin B,, (Barker et al., 1958). The reaction is freely reversible and involves a transfer of a hydrogen from the C-4 position of L-glutamate to the C-3 position, and a migration of the glycine residue to the C-4 position to form threo-@-methyl-Laspartate. No free intermediates have been detected (Barker et al., 1964) and there is no incorporation of deuterium from the solvent into the products (Iodice and Barker, 1963). Two distinct protein components, E (Suzuki and Barker, 1966) and S (Switzer and Barker, 1967), have been purified and both are essential for glutamate mutase activity. 2. Methylmalonyl-CoA Mutase
This reaction is very similar to the glutamate mutase reaction in that both a hydrogen transfer and a cleavage of a carbon-to-carbon bond with an alkyl group migration occur. A hydrogen moves from the C-3 position of succinyl CoA to the C-2 position while the CoA thioester carboxyl group migrates to the C-3 position (H. G. Wood et al., 1964; Eggerer et al., 1960). In this reaction, also, free intermediates have not been detected and there is no incorporation of hydrogen from the solvent into the products.
3. Diol Dehydrase Reaction Abeles and Lee (1961) first described this reaction in Aerobacter aerogenes. The enzyme catalyzes an irreversible conversion of either ethylene glycol or propylene glycol (propane-l,2-diol) to acetaldehyde or propionaldehyde, respectively. Hydrogen migrates from the C-1 position to the C-2 position and there is no exchange with hydrogen from the
METABOLIC ROLE O F VITAMIN Biz
iiq
399
1 . Glutamate mutase
LI~~--C-COOH
2.
FH.H HC -C-COOH I
-
/
NHz
COOH
Methylmalonyl- CoA m u t a s e
0 H II HC-C-SCOA COOH
COOH
3. Glycal dehydrase H
H HCH
I
HC=O 4.
Ribonucleotide r e d u c t a s e
LoJ
Base-C-C-C-C-C-0-P-P-P
-
H
lea
H Base-C-C-C-C-C-0-P-P-P H I H H OH
&HhHH
5. Ethanolamine deaminase H HC-NH,
=
I
HC-OH H 6. 8-Lysine
-
H
HTH HC=O
f
NH,
3,5-Diaminohexanoic acid
0 H H H H H l l HC-CC-C-C-C-C-OH I H H NH, I H NH2
-
0 H H H H H l l HC-C-C-C-C-C-OH H I H l H NH, NH,
FIG.2. BIa-Coensyme-dependentreactions.
solvent (Brownstein and Abeles, 1961). It is of interest to note that although an alkyl migration does not occur in this reaction (unlike the mutase reactions), Retey e t al. (1966) have shown that the oxygen atom on the C-2 position of propane-1,2-diol moves to the C-1 position of the propionaldehyde that is formed. Both a hemiacetal form of lactaldehyde
400
HERBERT WEISSIUCH AND ROBERT T. TAYLOR
and the hydrated form of the aldehyde product have been postulated by these authors to be intermediates in the conversion of propane-l,2-diol to propionaldehyde (Eq. 4 ) . H
-+
I HC-OH I
COB^
HCOH
I
CH3
--t
HC-OH
H
1;. I
OH
I
CHO
HC-OH -HCH
H-CoB12 CHI
I
-HzO
-CH2
AH3
I
(4)
CH,
Smiley and Sobolov (1962) have reported a reaction similar to the glycol dehydrase reaction using glycerol as a substrate. 4. Ribonucleotide Reductase Reaction
This reaction was first described by Blakley and Barker in extracts of Lactobacillus leichmunii (Blakley and Barker, 1964) and i t explains a t the enzymatic level the known ability of deoxyriboncleosides to alleviate the vitamin B,, growth requirement of this organism (Kitay et al., 1949). L. leichmanii ribonucleotide reductase requires a Blz-coenzyme and utilizes only nucleoside triphosphates (Abrams, 1965; Blakley et al., 1965; Blakley, 1966; Beck et ul., 196613) as opposed to the ribonucleotide reductase in Escherichia coli, which does not require a cobamide and is specific for nucleoside diphosphates (Reichard, 1962). A L. Zeichnzanii thioprotein reducing system (Orr and Vitols, 1966) similar to the thioredoxin system in E. coli (Moore and Reichard, 1963) normally functions in the cobamide-dependent reaction, although reduced lipoic acid alone can serve as a source of hydrogen in this system. Therefore, this reaction, unlike all the other BIZ-coenzyme-dependent reactions in Fig. 2, involves a net reduction with the incorporation of hydrogen from the solvent into the product. Studies on the mechanism of the hydrogen incorporation (Gottesman and Beck, 1966; Blakley et al., 1966) have shown that the solvent hydrogen is incorporated into the C-2’ position of the product, with retention of configuration (Batterham et al., 1967), suggesting that the hydroxyl group in the C-2’ position is displaced by a hydride ion via a stereospecific SN, mechanism. It should be noted that B,,-coenzyme-dependent enzymes show no apparent uniformity with regard to the stereochemistry of the reactions that they catalyze. Thus, in the glutamate mutase (Sprecher et al., 1966b) and glycol dehydrase (Zagalak et al., 1966) reactions inversion of substrate configuration occurs; whereas, in the methylmalonyl-CoA mutase (Sprecher et al., 1966a) and ribonucleotide reductase (Batterham e t al., 1967) reactions the configuration of the substrate is retained.
METABOLIC ROLE O F VITAMIN Biz
401
5. Ethanolamine Dehydrase Reaction This reaction was first reported by Bradbeer (1965) and has been studied recently by Babior (1967). Although detailed studies on the mechanism of this reaction have not been reported, it may be analogous to the glycol dehydrase reaction except that a deamination instead of a dehydration occurs. Again there is no incorporation of deuterium from the solvent into acetaldehyde (Babior, 1967). 6. Conversion of p-Lysine to 3,5-Diaminohexanoic Acid Stadtman showed that two strains of clostridia were able to ferment lysine to acetate, butyrate, and ammonia (Stadtman, 1963). Indirect experiments suggested a role of a cobamide coenzyme in this pathway. Studies by Costilow et al. (1966) identified 3,6-diaminohexanoate (Plysine) as the first product of lysine fermentation; it is formed in a pyridoxal phosphate-dependent reaction. The subsequent conversion of P-lysine to 3,5-diaminohexanoic acid requires 5'-deoxyadenosyl-B12 (Stadtman and Renz, 1967; Bray and Stadtman, 1968) and can be viewed as a migration of the amino group on C-6 to C-5 with a simultaneous transfer of hydrogen from C-5 to (2-6.
Iv. MECHANISMO F
REACTIONS It is now apparent that all the reactions shown in Fig. 2 involve a TH E 5'-DEOXYADENOSYL-B12-DEPENDENT
transfer of hydrogen. With the exception of the ribonucleotide reductase reaction, the hydrogen is transferred to an adjacent carbon with a migration in the reverse direction of a group (oxygen, alkyl, etc.) to the carbon which has lost its hydrogen. In the ribonucleotide reductase system (described in detail elsewhere in this volume) a hydrogen is transferred from the reducing agent into the product. The carrier of the hydrogen in the glycol dehydrase reaction has received the most attention and the role of deoxyadenosyl-B,, in this process is now well established. Abeles and Zagalak (1966) first demonstrated that hydrogen transfer in the glycol dehydrase reaction is not a direct intramolecular transfer despite the failure to incorporate hydrogen from the solvent into the products. In these studies, a radioactive substrate (l-3H-propane-1,2-diol) and a nonradioactive substrate (ethylene glycol) were simultaneously decomposed by the enzyme and radioactive acetaldehyde (3Hon C-2) was formed along with the expected radioactive product, propionaldehyde (3H on C-2). The ability to form a radioactive product from a nonradioactive substrate in these experiments indicated that the radioactive hydrogen from propane-l,2-diol must have been transferred to a position on the enzyme-coenzyme compIex which con-
402
HERBERT WEISSBACH AND ROBERT T. TAYLOR
tained a t least 2 equivalent hydrogens. Removal of a nonradioactive hydrogen from the enzyme-coenzyme complex would leave 3H on the enzyme-coenzyme complex which could subsequently be transferred to a molecule of unlabeled ethylene glycol yielding 3H-acetaldehyde. Abeles and Frey (1966) and Frey et al. (1967a) showed that the coenzyme was acting as a hydrogen carrier in the reaction by isolating radioactive coenzyme after incubating the enzyme with l-3H-propane-1,2-dio1. The isolated radioactive coenzyme could transfer its tritium to product in a subsequent reaction using an unlabeled substrate. I n other confirmatory experiments, chemically synthesized coenzyme, labeled with 3Hin the C-5’ position, transferred all its tritium t o propionaldehyde in the enzymatic oxidoreduction of unlabeled propane-l,2-diol (Frey and Aheles, 1966; Frey et al., 1967a). I n the glycol dehydrase reaction only about 1% of the l-3H-propane-1,2-dio1 is converted to propionaldehyde by the abstraction of a hydrogen from the C-1 position followed by its return to the C-2 position of the same molecule (Frey et al., 1967a). The essential role of the hydrogen a t the C-5’ position of the coenzyme has been further demonstrated by the studies of Retey and Arigoni (1966). Csing coenzyme labeled with 3H on the C-5’ position by means of the diol dehydrase reaction, they were able to demonstrate the transfer of tritium to products during catalysis of the methylmalonyl-CoA mutase reaction. The ribonucleotide reductase reaction is atypical, however, compared to the other reactions shown in Fig. 2 in that tritium on the C-5’ of chemically synthesized BIZ-coenzyme does not become incorporated into the deoxyribonucleotide product. Instead, tritium was transferred quantitatively from the (3-5’ position into water in a reaction system that contained ribonucleotide enzyme, substrate, and a reducing agent (Beck et al., 1966a). It has been suggested (Beck e t al., 1966a), therefore, that the enzyme catalyzes a reversible hydrogen transfer between the reducing agent (reduced lipoate) and the coenzyme-enzyme complex so that rapid equilibration of the hydrogen on the reducing agent with water could explain the results that were obtained. This was substantiated when Hogenkamp et al. (1967) observed the exchange of tritium between water and the C-5’ position even when the substrate ribonucleotides were replaced by any one of the several deoxyribonucleotides, especially deoxyGTP. Although the incubation components needed for solvent hydrogencoenzyme exchange closely mimic those needed for the overall reduction (Fig. 2) (Beck et al., 1966a; Hogenkamp et aE., 1967; Abeles and Beck, 1967) and the rate of hydrogen exchange is comparable to the overall rate of reduction (Hogenkamp et al., 1967), it is still not definitely established that the coenzyme actually functions as an intermediate hydrogen carrier in this system. It is particularly important to note t ha t in both the glycol dehydrase
METABOLIC ROLE O F VITAMIN Biz
403
and the ribonucleotide reductase systems both hydrogens on the (2-5’ position of the B,,-coenzyme-enzyme complex arc labilized (Frey et al., 1967b) during catalysis. Yet, these two (3-5’ hydrogens are not sterically equivalent on the unbound, free B,,-coenzyme-containing tritium in the C-5’ position. This was shown conclusively when both isomers of BIZcoenzyme-C-5’ (3H) were prepared and each transferred all its tritium to propionaldehyde in the glycol dehydrase system. When the ribonucleotide reductase reaction was carried out in pure deuterium oxide, 1.6 atoms of deuterium per molecule were incorporated a t the C-5’ position of the coenzyme. These findings establish that a t some point in B,,-coenzymedependent reactions the two C-5’ hydrogens became sterically equivalent, perhaps by a cleavage of the carbon-cobalt bond atom a t the C-5’ position. I n this connection Tamao et al. (1967) have attributed the reduced activity of 10-C1-BIZ-coenzyme (ie., chloro on the C-10 of the corrin nucleus) in the glycol dehydrase system to a polarization of the carboncobalt bond, the electrons being more attracted to the cobalt. They have consequently suggested that a carbon-cobalt bond cleavage occurs to give a 5’-deoxyadenosyl carbanion ( eCH, - R) . It must be concluded that in all the B,,-coenzyme-dependent reactions, hydrogen from either the substrate or a reducing agent (as in the case of the ribonucleotide reductase reaction) is transferred to the C-5’ position of the coenzyme bound to the enzyme. With the exception of the ribonucleotide reductase reaction, the hydrogen in the enzyme-coenzyme complex is subsequently transferred to the product without equilibrating with hydrogen of the solvent.
V. METHYLTRANSFERASE REACTIONS Three cobamide-dependent reactions are now known which do not require 5‘-deoxyadenosyl-B,,. Instead, a tightly bound cobamide prosthetic group is functional, and the enzymes involved catalyze methyl group transfer reactions (Fig. 3) in which methyl-BIZ probably serves as a protein-bound intermediate. Included in this group of reactions are the syntheses of methionine, methane, and acetate. I n all cases, N5-methylH,-folate, as well as methyl-B,,, can serve as the direct methyl donor, although the folate derivative is probably the natural substrate.
1. Methionine Synthesis This reaction appears to be the simplest of the cobamide-dependent methyltransferase reactions and has been investigated in the greatest detail. The studies by Woods and co-workers (Guest e t al., 1960, 1964; Kisliuk and Woods, 1960; Foster et nl., 1964) as well as Buchanan and collaborators (Larrabee e t al., 1961; Hatch et al., 1961; Takeyama et al., 1961) contributed largely to the understanding of the requirements and
404
HERBERT WEISSITACH AXD ROBERT T. TAYLOR 1.
Methionine synthesls N5-Methyl-H,-Folate
2.
+
Hornocysteine _c Methionine
+
H,-Folate
Methane synthesis N5-Methyl-H,-Folate -Methane
3. Acetate synthesis NS-Methyl-H,-Folate
+
C0,--Acetate
7
Methionine
CH,
2‘0’
I’
‘
Methyl- B
l
Methane q Acetate
FIG.3. Cobamide-dependent methyltransferme reactions.
the general characteristics of the reaction in Escherichia coli. The reaction, shown below, involves a methyl group transfer from N5-methyl-H,folate (Larrabee et a;., 1961) t.o homocysteine to form methionine and H,-folate (Eq. 5.) N6-Methyl-Ha-folate
AMe Protein > Methionine reducing system
+ Homocysteine
+ H4-Folate
(5)
A cobamide-containing protein (Guest et al., 1960), a reducing system (Guest et al., 1960; Hatch et al., 1961), and catalytic levels of S-adenosylmethionine (AMe) (Mangum and Scrimgeour, 1962; Rosenthal and Buchanan, 1963; Foster et al., 1964; Weissbach et al., 1963) are essential. The observation by Guest et al. (1962) that the enzyme also catalyzed the transfer of a methyl group from methyl-B,, to homocysteine focused attention on the possibility that methyl-B,, might be an enzyme-bound intermediate in methyl group transfer from Nj-methyl-H,-folate t o homocysteine. I n the reaction sequence suggested below, the folate derivative methylates a reduced cobamide species on the enzyme to form a methyl-B,,-enzyme and H,-folate. Then a subsequent transfer of the methyl group to homocysteine occurs to yield methionine and to regenerate a reduced B,,-enzyme (Eq. 6 ) . H4-Folate
N6-Methyl-H4-folate
+\I
/I
/
Co+
\
Enzyme t
AMe
+
CHI
\ I /’ Co /I \
Enzyme
Komocysteine
I
> Methionine
(6)
METABOLIC ROLE O F VITAMIN Biz
405
In this two-step reaction sequence, AMe is pictured as being required only for alkylation of the enzyme by N5-methyl-H,-folate. Extensively purified preparations of the enzyme have been obtained from extracts of E. coli (Takeyama and Buchanan, 1961; Taylor and Weissbach, 1967a). The B,, enzyme in such preparations is salmoncolored and displays an absorption spectrum (475 mp maximum) that closely resembles the spectrum of B,,, (Taylor and Weissbach, 1967a) or a B1,-thiol complex (Dolphin and Johnson, 1963). These spectral similarities and the fact that sulfito-B,, is obtained (Takeyama and Buchanan, 1961; Ertel e t al., 1968) upon alcohol extraction of the B,,-protein suggest that the cobamide may be linked to an SH group on the enzyme. Studies utilizing millimicromole amounts of enzyme and either radioactive N5-methyl-14C-H,-folate or methyl-I4C-AMe have shown that both compounds react with the bound cobalamin to yield a methyl-Blzenzyme (Taylor and Weissbach, 196713, 1968a). A reducing system is required for both methyl donors to alkylate the bound cobamide, and in addition, AMe is needed for methyl group transfer from N5-methyl-H4folate to the enzyme-bound cobamide. Of interest was the finding that a methyl-14C-B,, enzyme was stable to light (as measured by the release of volatile I4C or radioactive formaldehyde) until the protein was acidified to below pH 2.5 (Taylor and Weissbach, 1968a). Definitive proof that a methyl-B12-enzyme was formed in these experiments was obtained by ethanol extraction of the bound cobamide followed by identification of the radioactive chromophore as methyl-"C-B,,. If the methyl-B1,enzyme is isolated by procedures that do not cause denaturation (e.g., Sephadex gel filtration), it is active catalytically, and it can transfer its methyl-I4C group to homocysteine to form methyl-*4C-methionine (Taylor and Weissbach, 1 9 6 7 ~ )This . reaction proceeds to completion in air without the requirement of either a reducing system or exogenous AMe. Essentially, these foregoing observations have been made recently by Stavrianopoulos and Jaenicke (1967)) who also used highly purified E. coli transmethylase. Recent studies on the time course of methylation of the bound cobamide in the presence of both AMe and N5-methyl-H,-folate have shown that AMe rapidly methylates the enzyme-bound cobamide first and methyl groups from the folate donor subsequently replace the methyl groups on the cobamide which originated from AMe (Taylor and Weissbach, 196813). I n this regard, it has also been possible t o show that an enzyme methylated with AMe, and then reisolated, will catalyze limited methyl group transfer from N5-methyl-H,-folate to homocysteine in the absence of exogenous AMe and a reducing system (Taylor and Weissbach, 196%). This "priming" action occurs only under conditions in which
406
HERBERT WEISSHACH AND ROBERT T. TAYLOR
AMe is able to methylate the cobamide on the enzyme and has also been accomplished with methyl iodide. These observations suggest that AMe may function in its normal manner as a methyl donor but methylate only the initial molecules of BIZenzyme. The bound cobamide attached initially to the enzyme is present in an inactive form, perhaps linked to a sulfur on the protein. AMe or an efficient chemical methylating agent such as methyl iodide (Taylor and Weissbach, 1967d) might react with the enzyme-bound cobamide to form a methyl-B,,-enzyme; hence, the first methyl group on the enzyme would arise from AMe. Transfer of this methyl group to homocysteine as a carboiiium ion would yield a reduced cobamide species (Cot) on the enzyme which could rapidly accept subsequent methyl groups only from N5-methyl-H,-folate. N5-Methyl-H,-folate would then continue to supply methyl groups for methionine synthesis until the cobamide on the enzyme was oxidized back to its original inactive form. Activation, or priming, would thus be accomplished by AMe in a catalytic manner. 2. Methane Formation
A cobamide derivative was implicated in methane formation after the initial observation by Blaylock and Stadtman that methyl-B,, was converted to methane by extracts of Methanosarcina barkeri (Blaylock and Stadtman, 1963). Additional studies by Blaylock and Stadtman (1964), as well as by Wolin et al. (1963) using extracts of M e t h a n ~ b a ~ i l ~ u s omeliansfcii, have further suggested the participation of a protein-bound cobamide in methane formation. Direct evidence for the involvement of a cobamide containing enzyme has come from the studies of J. M. Wood and Wolfe (1966a), who isolated from M . omelianskii a methane-forming enzyme containing 5-hydroxybenzimidazolyl cobamide (factor 111). In addition t o a reducing system, ATP is required for methane formation from methyl-B,, (Wolin et al., 1963), N5-methyl-H,-folate (J. M. Wood et al., 1965), and methyl-B,, analogs (J. M. Wood et al., 1966). J. M. Wood and Wolfe (1966b) reported that 1 mole of ATP was decomposed per mole of methane formed. Blaylock and Stadtman (1966) have shown that methanol, which is a substrate for methane formation, can donate, in the presence of ATP and a reducing system, a methyl group to B,,, forming methyl-B,,. While the available evidence suggests that a free methyl cobamide is not an intermediate in methane formation, an enzyme-bound methyl cobamide (as pictured in the methionine reaction) is probably involved. The enzymes that catalyze methionine and methane formation can both be inhibited by reacting the protein-bound chromophore with propyl iodide to form a propyl-cobamide enzyme (Brot and Weissbach, 1965; J. M. Wood and Wolfe, 1966a; Taylor and Weissbach,
407
METABOLIC ROLE O F VITAMIN Biz
1967e). Restoration of catalytic activity to a propylated enzyme by exposure to visible light strongly suggests that the cobalt atom is directly involved in these enzymatic processes and that a common reaction mechanism exists. It is noteworthy, however, that cobalt methyl derivatives have not been found in extracts of methane-forming organisms (Lezius and Barker, 1965). 3. Acetate Synthesis
Barker and Kamen (1945) first demonstrated that Clostridium thermoaceticum utilized COz for the total synthesis of acetate. Poston et d. (1964) provided evidence that this reaction involved a cobamide derivative. The authors observed that the formation of the methyl group of acetate from CO, in cell-free extracts was inhibited by intrinsic factor and that methyl-B,, could serve as a methyl donor for the methyl group of acetate. The exact sequence by which CO, is incorporated into acetate and the role of cobamide derivatives in this process is still unknown. Irion and Ljungdahl (1965) have isolated a wide variety of corrinoid compounds from C . thermoaceticum including methyl and deoxyadenosyl derivatives of cobyric acid and 5-methoxybenzimidazolyl cobamide (factor IIIm). Exposure of the cells to 14C0, resulted in the rapid labeling of cobalt methyl groups in methylcobyric acid, methyl factor IIIm, and traces of carboxymethyl corrinoid (Ljungdahl et aZ., 1965). These and additional isotopic experiments (Ljungdahl et al., 1967) indicate that a protein-bound cobalt methy1J4C corrinoid is formed upon incubation with 14C0, and that this methyl-14C corrinoid is then converted to a proteinbound, cobalt carboxymethyl derivative which can be reductively cleaved to acetate. Two protein fractions and ferredoxin have been separated from extracts of C . thermoaceticum (Poston and Stadtman, 1967) and are required in combination with CoA and pyruvate to convert methyl-I4Ccobalamin to radioactive acetate. A pathway summarizing these experiments has been postulated by Ljungdahl et al. (1966) and is shown in Eq. (7).
I
1
auction
CHICOOH
Thus, in all the methyl transferase reactions, a methyl-B1, protein is currently considered to be an intermediate.
408
HERBERT WEISSE'ACH
AND ROBERT T. TAYLOR
VI. ROLEOF VITAMINB,
IN
ANIMAL METABOLISM
Studies with pernicious anemia patients have suggested that vitamin B,, is required primarily for DNA synthesis which is essential for normal development of the mature red blood cell in the bone marrow. Considering the reactions that were described in the preceding sections, attention is immediately focused on the participation of cobamide derivatives in ribonucleotide reduction and the formation of methionine, two possible enzymatic sites of BIZaction that would affect DNA synthesis. Although only limited data are available (Moore and Hurlbert, 1962), there is, as yet, no evidence in support of the view that ribonucleotide reduction in animal systems requires a cobamide derivative. I n fact, of the known reactions that require 5'-deoxyadenosyl-B,, (Fig. 2) , only the methylmalonyl-CoA mutase reaction has been demonstrated in animal tissues (Lengyel et al., 1960). Of the methyl transferase reactions shown in Fig. 3, there is substantial evidence that the synthesis of methionine requires a cobamide derivative in animal tissues (Loughlin et al., 1964; Dickerman e t al., 1964). In addition, it is well established that there is a nutritional interrelationship among folic acid, vitamin B,,, and onecarbon metabolism (Bennett et al., 1951; Machlin et al., 1952; Sunde et al., 1951; Fox et al., 1956, 1961; Silverman and Pitney, 1959). A summary of various tetrahydrofolic acid derivatives which depicts their role in one-carbon metabolism is shown in Fig. 4. It is seen that tetrahydrofolate is directly involved in the synthesis of purines, thymidylate, and methionine. I n vitamin B12deficiency, the rate of methionine synthesis might N"'- Formyl-Folate-H,
Histidine -FIGLU Serine Folate-H,
N5,lo-Methylene-Folate-H,
N5-Methyl- Folate-H,
FIG.4. Metabolism of folic acid derivatives.
METABOLIC ROLE O F VITAMIN Biz
409
be diminished and the ability to regenerate H,-folate from N5-methyl-Hafolate would be impaired. If the synthesis of methionine from homocysteine is a major means by which H,-folate is regenerated in the body, a deficiency of vitamin B,, would markedly alter folate metabolism and result in an accumulation of N5-methyl-H,-folate a t the expense of H4folate and its methylene and methenyl derivatives. Direct determination of folate compounds in animals on vitamin B,,-deficient diets have confirmed that the partition of folk acid derivatives is changed in vitamin B,, deficiency (Norohna and Silverman, 1961). I n addition, the increased excretion of formiminoglutamic acid (FIGLU) observed in vitamin BIZ deficiency (Fox e t al., 1961 ; Silverman and Pitney, 1959) is presumably caused by a deficiency of H,-folate. Based on these data, i t is plausible that many of the effects of vitamin B,, deficiency in bone marrow are related to an altered metabolism of €I,-folate. I n contrast, the central nervous system lesion seen in pernicious anemia could be related to the methylmalonyl-CoA mutase reaction. REFERENCES Abeles, R. H., and Beck, W. S. (1967). J . Biol. Chem. 242, 3589. Abeles, R. H., and Frey, P. A. (1966). Federation Proc. 25, 1639. Abeles, R. H., and Lee, H. A. (1961). J . B i d . Chem. 236, 2347. Abeles, R. H., and Zagalak, B. (1966). J . Biol. Chem. 241, 1245. Abrams, R. (1965). J . Biol. Chem. 240, PC3697. Babior, B. (1%7). Federation Proc. 26, 344. Barker, H. A., and Kamen, M. D. (1945). Proc. Natl. Acad. Sci. U S . 31, 219. Barker, H. A., Weissbach, H., and Smyth, R. D. (1958). Proc. Natl. Acad. Sci. U.S. 44, 1093.
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