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Enzymatic Reduction of Ribonucleotides Biosynthesis Pathway of Deoxyribonucleotides.

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15. 34
Enzymatic Reduction of Ribonucleotides :
Biosynthesis Pathway of Deoxyribonucleotides
By Hartrnut Follrnann[*]
Ribonucleotide reductases are enzymes that synthesize the deoxyribonucleotides required for
the replication of DNA in dividing cells. They thus have a key function for the growth of
microorganisms and of all plant and animal tissues. The enzymes reduce all four purine
and pyrimidine ribonucleotides (as the 5'-diphosphates or triphosphates) with direct substitution
of the ''-hydroxyl group by hydrogen. The physiological reducing agents are the mercapto
groups of thioredoxins, a group of small proteins, which are regenerated from the oxidized
form by NADPH-dependent thioredoxin reductases. There are two known types of ribonucleotide
reductases ( I and II), which catalyze hydrogen transfer with the aid of protein-bound iron
ions or of 5'-deoxyadenosylcobalamin (coenzyme B l z); free radIca!s can be detected in both
cases. The enzymes are regulated by effector nucleotides. There may exist a homeostatic
mechanism, which guarantees the supply of DNA precursors to the cell.
1. Deoxyribonucleotides as Building Units of DNA
In a bacterium or growing cell, before new deoxyribonucleic
acid (DNA),the macromolecular carrier of the genetic information in the chromosomes, can be synthesized during cell divi[*] Prof. Dr. H. Follmann
Fachbereich C'hemie der Univcrsitiit
Arheitsgruppe Biochcmie
355 M a r b u r p Lahn. Lahnburge ( G e r m a n y )
[*"I T h e S'-phosphatcs of thc rihonucleosides adenosine IA). cytidine I<').
guanosinc (GI. a n d iiridinc IL') a n d o f the deoxyrihonticleosides deoxyadenobinc IdAl, dcoxycytidlne (dC). deoxyguanosine (dG). and thymidine (dT) a r e
denoted in thc usual manner by the abbreviations - D P (diphosphate) a n d
-TP (triphosphateJ: I d J N is a n tinspecified (droxy)ribonu~leoside.D N A :
dcoryrihonucleic dcid. R N A . rihonucleic acid, FAD: flavin adenine dinuclcotidc. N A D P H and N A D P . reduced and oxidized nicotinamide adenine dinucIeotidu phosphate rcspecliwly. F o r thu s t r t i ~ t t ~ r eofs these comnotinds cf
M.: S a ~ ~ i nAngew.
/ ~ ~ . Chenl. 8.5, 680 (1973): Angew. Chem. Intcrnat. Edit
/2. S Y i (19731
A n y r w . Chrm. inrernat. Edit.
I Voi. 13 I i974) / No. 9
sion, its monomeric building units must be available in the
cell as substrates of the replicating enzymes. This condition
seems obvious, but among the numerous processes in the
synthesis of nucleic acidsfi1(Fig. I), surprisingly little is known
about the formation of the four deoxyribonucleotides (step
b), in contrast with the very extensively studied biosynthesis
of the purine and pyrimidine ribonucleotides (step a). Similarly,
the polymerization of deoxyribonucleotides (step c) iri r i m
has not yet been fully elucidated in all important details.
Though the 5'-triphosphates of deoxyadenosine, deoxycytidine, deoxyguanosine, and thymidine (dATP. dCTP, dGTP,
and dTTP["I) are generally considered the immediate precursors of DNA, an enzymatic polymerization of the corresponding 5'-diphosphates is also known[", and a DNA replication
at the level of 5'-monophosphates activated in some other
way has been suggested13!
All these deoxyribonucleotides are present in the cell only
in minute steady-state concentrations. They can hardly be
detected by direct analysis in the presence of the chemically
similar ribonucleotides, whose concentrations are higher by
amino acids, ammonia,
f o r m a t e , carbon dioxide, A T P
2 ' -deoxyribonucleotide s
Fig. I. Dependence of the DNA replication (step c ) on the purine and
pyrimidine biosynihesis (step a ) and the ribonucleotide reduction (step b)
in the course of the r / c ~ - n o i % nucleic acid biosynthesis.
a factor of 10 to 100. It is only
that the deoxyribonucleoside triphosphates present in a tissue extract can
be quantitatively determined in amounts of less than 1 pmole
with theaid of DNA polymerase I and synthetic polynucleotide
templates in citro. Pools of the order of 10-100pmole of
dNTP per 10' cells were found in cell cultures of mice['.']
and of hamsters1s.91andin ascites tumor
these quantities being distributed between the cell nucleus and the cytoplasm" 'I. They exhibit considerable growth-dependent variations['.'"*], but they can only sustain DNA replication for
about 1-5min in any case. Only oocytes such as those of
the frog Xenopus laecis appear to be better supplied with DNA
precursors, since the pool of 12 pmole of dNTP per cell found
is sufficient for the synthesis of the DNA of more
than 2000 new cell nuclei.
Thereare two possible routes for replenishment of the deoxyribonucleotide pools, i. P . reutilization of DNA fragments such
as thymidine from the catabolic metabolism or supplied from
outside, or complete synthesis of new rnaterial[l3'. The first
route ("salvage pathway") is essential for auxotrophic microorganisms; it requires a series of inducible enzymes such as
nucleoside deoxyribosyl transferases, nucleoside phosphorylases, and nucleoside kinases, which can separate the bases,
sugars, and phosphate residues of the nucleotides from one
another and recombine them.
Since all other organisms synthesize DNA without the supply
of deoxyribonucleotides and make practically no use of nutritive nucleic acids, they must all be capable of de-noco synthesis.
The pentose component of the nucleic acids is obtained here
from the oxidative and non-oxidative carbohydrate metabolism of the cell, as can be shown e.g. by the isotope content
of the RNA and DNA when Eschcjrichia coli is cultured on
'MO-labeledglucose or fructosecJ4].In none of these pathways
is free 2-deoxyribose formed and then incorporated into nucleotides; the enzyme deoxyribose aldolase that is in principle
able to synthesize this sugar from glyceraldehyde-3-phosphate
and acetaldehyde" 'I probably catalyzes only the degradation
of 2-deoxyribose-5-phosphate in vino1'61.The biosynthesis of
all nucleotides proceeds instead from ribose-5-phosphate. with
construction of the heterocyclic bases (Fig. 1) to form ribonucleotides, from which the deoxyribonucleotides are formed
5 70
in a further reaction, with retention of the N-glycoside linkage
between the sugar and the base. This relationship was first
demonstrated by Reichard in 1950 in the utilization of I 'Nlabeled cytidine (but not cytosine) for RNA and DNA in the
metabolism of the living rat"']. The conversion of *4C-Iabeled
ribonucleosides into deoxyribonucleotides was also detected
first in microorganism cultures" 81 and later in cell-free extracts
(e.g.from E. coIi["]).
Enzymes that reduce ribonucleoside-5'-phosphates with replacement of the 2'-hydroxyl group by hydrogen to form 2'-deoxyribonucleotides are known as ribonucleotide reductasesf201.
The reducing agents in every known case are dithiols, which
are converted into the disulfide and regenerated in a further
enzyme reaction. There are only few reviews of these reactions[i.13~z'.221.
The present report deals with our current
knowledge of the occurrence and the structure of ribonucleotide reductases, their reaction mechanism, and their unusual
regulation phenomena. Numerous investigations during the
past few years have shown that these enzymes, to which little
attention had previously been given, are of outstanding importance to the development of all life forms.
2. Ribonucleotide Reductases
2.1. Occurrence, Isolation, and Detection
Ribonucleotide reductases, as inducible enzymes very closely
connected with cell proliferation, are most easily found in
rapidly growing cell cultures and tissues; great difficulty is
encountered in their detection in resting tissues. Bacteriophages,
microorganisms in the exponential growth phase, germinating
plant seeds, embryonic or regenerating animal organs and
tumors are typical materials for the isolation of ribonucleotide
reductases (Table I). The majority of the known enzymes
come from bacteria and mammalian tissues, whereas the reduction of ribonucleotides in plants, for example, has not yet
been very extensively studied. The reductases of Escherichia
coli, of Lactobaciilus leichmannii, and from Novikoff tumors,
which have been under investigation for some time by the
research groups of Reichard (Stockholm),Blaklry (Iowa City),
and Moore (Houston), exhibit a number of properties that
are typical of a11 enzymes, but the deoxyribonucleotide synthesis as a whole exhibits many species-specific peculiarities
in most organisms.
The very limited knowledge of the reduction of ribonucleotides
that existed until recently bears witness to the difficulties
of dealing with these enzymes. They always occur in small
quantities and are often unstable proteins, so that they are
not easy to purify by conventional methods (e.g.from yeast[321
or wheat'331). A successful use of affinity chromatography
with insolubilized coenzyme Bl 2 [ 5 1 1 or dATPCs2]
for the purification of the ribonucleotide reductases from L. kichmannii,
E. cob, and the bacteriophage T4 promises progress in this
Ribonucieotide reductases are also much more difficult to
determine quantitatively than most other enzymes. There is
no fast routineassay for the simple reaction ( 1 ) that is catalyzed
by them. Themost common method makes use of a radioactive
substrate such as [5-3H]CDP, the resulting labeled deoxyribonucleotide being determined after separation by paper,
column['3. "]. or thin layer chromatography[541;this test is
Angew. Chum. infernat. Edir. J Vol. 13 (1974)
No. 9
S iih\trdtes
N D I’
Fc. ATP. Mg
Fc. ATP. IMs)
cocnrymc BI!
coenzyme BI
cociiiymc B ,
coenzyme B,
Mp. ATI’
Fc. AT[’
Mg. ATI’
Fc. ATP. Mp
Fe. ATP, Mg
Fc. ATI’, Mg
subject to interference by other nucleotide-transforming
enzymes, particularly in crude extracts. A less sensitive method
is the colorimetric determination ofdeoxyribose with diphenylamine ; in this case, the deoxyribonucleotide formed must
first be hydrolyzed with acid[551.A method that is also suitable
for kinetic measurements simulates the physiological process,
the reaction ( 1 ) being coupled with the regeneration of the
dithiol thioredoxin by thioredoxin reductase [reaction (2)],
and the NADPH consumption being followed spectrometrially'^''; however, purified enzymes and thioredoxin of suitable specificity are necessary for this enzyme determination.
Finally, the coenzyme B 2-dependent ribonucleotide reductases (enzymes of type I1 in Table 1 ) can be determined by
the liberation of tritium from [5’-3H]-5’-deoxyadenosylcobalamin (coenzyme Biz, see below) during the reduction. The
radioactive water formed is sublimed out of the reaction solution for this determination‘”. 571.
Angel*.. Chrm. intmnat. Edit. 1 Vol. 13 (1974)
1 No. 9
2.2. Specificity of the Enzymes
All purified ribonucleotide reductases catalyze the reduction
of the four natural ribonucleotides (and also of ribonucleotides
with non-natural bases) by the same enzyme protein; there
is no indication that the other systems contain several enzymes
that are specific for individual nucleotides. All the ribonucleotide reductases are also alike in that they accept low molecular
weight dithiols such as dithioerythritol or reduced lipoic acid
as reducing agents in ritro, and they require the 5’-diphosphates
or 5’-triphosphates ofthe ribonucleosides as substrates. Reduction at the 5’-monophosphate level does not seem to occur,
though nucleotide kinases may give the incorrect impression
that it doesoccur in unpurified extracts. However, the ribonucleotide reductases must be divided into two very different
groups according to cofactor requirement and protein nature.
2.2.I . Ribonucleoside Diphosphate Reductases (EC
Enzymes of type I (Table I), represented by that from E.
coli, are found in microorganisms, plants, and animals as
high as man. So far as is known, they always reduce the
diphosphates ADP, CDP, GDP, and UDP. A characteristic
of this group is that instead of catalyzing the redox reaction
(hydrogen transfer) between dithiol and ribose with the aid
of one of the conventional coenzymes, they contain proteinbound iron ions, which are essential for the activity of the
enzyme. The existence of such an enzyme is therefore often
recognized by the fact that reduction of ribonucleotides is
stimulated by low concentrations of iron salts or inhibited
by coniplexingagents for iron. The enzyme reaction is inhibited
particulary specifically by hydroxyurea[581and by 1 -formylisoquinoline t h i o s e m i c a r b a ~ o n e ' ~however,
this inhibition
is only partly reversible, and probably also leads to further
changes in the proteins. Many of these enzymes also require
magnesium ions and ATP (as an effector, and not as an energy
source) for their activity. It is not yet possible to say whether
this is true of all reductases of type I.
2.2.2. Ribonucleoside Triphosphate Reductases (EC
Type I1 (Table 1 ) comprises enzymes whose hydrogen transfer
reaction involves 5'-deoxyadenosylcobalamin. This coenzyme
form of vitamin 912 catalyzes several enzymatic hydrogen
shifts ria the 5'-methylene group of its adenosyl residue (cf.
Fig. 3)[601;its participation in the reduction of ribonucleotides
was first detected by Bhkky and Barker[251in a cell-free
extract of Lactobacilltrs. A number of enzymes can evidently
also use diphosphates as substrates instead of the triphosphates
ATP, CTP, GTP, and UTP. They are not greatly affected
by metal ions. Apart from the lower algae Euglena. the coenzyme
B1z-dependent ribonucleotide reductases seem to occur only
in prokaryotes (organisms without cell nuclei). Their distribution in many microorganisms has been investigated by the
very sensitive test with radioactive coenzyme128J,but there
is no regularity of occurrence here, even in closely related
species. Many attempts have been made to demonstrate the
participation of B12-dependent reductases in the DNA synthesis of mammals, particularly in the unbalanced DNA formation in the megaloblasts of the bone marrow in pernicious
anemia, but the action of vitamin 9, in these systems is definitely not connected with the reduction of r i b o n u ~ l e o t i d e s ( ~ ~ ~ .
2.3. Protein Structure
Only a few of the ribonucleotide reductases listed in Table
1 have been purified sufficiently for detailed analyses of their
protein structure and of the bonding of substrates and effectors.
No far-reaching comparisons are possible from a glance at
Table 2. Nevertheless, it can be seen that several iron-dependent reductases are large proteins with two dissimilar com-
ponents, each of which is inactive by itself, and which may
be regarded as a regulatory subunit and a catalytic subunit.
Thus in the case of the separated pure subunits of the enzyme
from E. colil"], protein B1 has at least four different kinds
of nucleotide binding sites1h51,whereas B2 binds two iron
atoms, but no nucleotides. Their enzymatically active I : I
complex is formed in the presence of magnesium ions and
the positive effector dTTP; binding of the inhibitor dATP
leads to an inactive, aggregated protein. The amino acid analysis and other properties of this enzyme system have been
The manner in which the iron atoms are bound is particularly
interesting in these proteins. In the E. coli protein 92, they
are inert to reducing and oxidizing agents and are not bonded
to inorganic sulfur (as in other "non-heme iron" compounds):
they can be removed from the protein by careful dialysis
against 8-hydroxyquinoline 5-sulfonate and imidazole, and
can be reconstituted to give a fully functional 9 2 by treatment
of the enzymatically inactive apoprotein with iron( 11) ascorbatel '1.
The U V absorption spectrum of the metalloprotein, like the
Mossbauer spectrum (recorded for the ["Fe]protein B2)ra71,
points to a close structural relationship with the hemerythrins;
in these oxygen-transporting proteins of marine worms and
brachiopods, there are two "high-spin" iron(ir1)ions in an antiferromagnetically coupled (i. e. as a whole diamagnetic) binuclear complex, whose protein ligands are unknown. The function of the iron in B2 is evidently not the transport of either
oxygen or electrons; it serves for the production and stabilization of an organic radical that is also detectable in the subunit
B2 and that is probably involved in the reduction (see Section
3.1.). The T4 phage enzymeL6']is very similar in all these properties to the enzyme from E. coli.
The coenzyme B l 2-dependent reductases seem to have somewhat simpler structures. The relatively small Lactobacillus
enzyme combines all the catalytic and regulatory binding
sites (at least four for the substrate, coenzyme, and effectors)
in a single polypeptide chain, which is not unusually
Its amino acid analysis and initial attempts to prepare large
protein fragments have been described; kinetic and mechanistic studies are particularly well advanced for this enzyme.
Table 2. Protein structure of purified ribonucleotide redtictases.
.......... ......
Source and purity
9.7 - - I 0 I
Separable by chromatography into B I (m.wt 160000.
2 peptide chains) and B 2 (m.wt. 78000. 2 pcptide
chains): B I binds nticlcotides, B 2 contains 2 atoms
of Fe (light abs. 360.410 nm): B I : B 2 complex
stabilized by Mg- in presence of dATP inactive dimcr
I Protein having 2 atoms of Fe (light abs. 360, 410 nm).
only slightly stabilized by Mg; denaturing electrophoresis
yields 2 pairs of peptide chains having m.wt. X0000 and
35000 resp.
Aggregated and inactivated in presencc of dATP
Separable by chromatography into 2 dissimilar proteins
S 1 and S 2, inactive when separated
2 Dissimilar proteins PI and P2
I Polypeptide chain ( ( a . 690 amino acids), incapable of
dissociation or aggregation
Activity maximum at 70 C,stablc tip t o XO C. effector
action only at elevated temperature (change of conformation?)
Phage T4
Rat liver I300 x )
Bone marrow (50 x )
6.6 S
Novikoff tumor 1 1 5 0 1~
L. leichmannii (homogeneous)
Thc,rrmrs X - l
.... ...
M.wt. and/or
Angew. Chem. intanat. Edit.
Vol. 13 (1974) f No. 9
2.4. Subcellular Localization
body temperature? Deoxygenations at high temperatures or
catalytic hydrogenations can hardly be cited as non-enzymatic
parallels for nucleotide molecules, and the only known chernical conversion of a ribonucleotide (AMP)into the corresponding deoxyribonucleotide by linkage of the 2'-hydroxyl group
and the adenine base followed by removal of sulfur from
the intermediate 8,2'-anhydroadenosine thioether [reaction
also does not proceed under physiological conditions.
The high specificity of the enzyme reaction for ribonucleotides
with an intact 2',3'-~is-diol groupingi"' can be interpreted
by multiple fixation of the substrates to the protein r:iu the
phosphate ions, the purine or pyrimidine base, and the 3'-hydroxyl group(cf. Fig.4), but an explanation ofthe redox reaction
itselfpresents bothchemistsand enzymologists with a problem
that is as difficult as it is interesting.
The localization of the ribonucleotide reductases in the cell
has not yet been systematically studied. They are generally
thought to be cytoplasmic enzymes, since little or no reductase
activity is found in cell nuclei, mitochondria, and microsomes
of rat liver[''' or of Novikoff tumorsihs1.There have recently
been indications that these tissues contain ribonucleotide
reductase as an enzyme complex together with other enzymes
ofthe DNA biosynthesis, i. e. thymidylate synthetase, thymidylate kinase, and DNA polymerase I I f h X ~ h YThese
activities sedimented in the ultracentrifuge with a postmicrosoma1 membrane fraction of unknown origin, and after breakdown of the membrane fragments they were visible in the
electron microscope as particles with a size of 8.5-12nm.
The membrane component does not seem to be essential
to the activity and cohesion of the enzymes. Similar material
with a high reductase activity has also been isolated from
thymus, spleen, and HeLa cells.
The fate of the atoms and groups involved has been so thoroughly studied in the enzymes from E. C a l i and L. Irichmunnii
with isotope-substituted model nucleotides and spectroscopic
techniques that, despite the very different protein structures
Fig. 2. Possible reaction pathways for the replacement of the 2'-hydroxyl group of ribose by hydrogen: a) direct
substitution, b) dehydration-hydrogenation, c ) oxidation-reduction, d) actwation by ATP. P = phosphate
residue, B =purine or pyrimidine base.
3. Mode of Action and Regulation
3.1. Reduction of the Hydroxyl Group
How is one of the two secondary hydroxyl groups of ribose
selectively reduced to a methylene group at pH=7 and at
Angew. Chem. internat. Edit. f Vol 13 (1974) f No. 9
and cofactors, it is conceivable that the modes of action of
these two bacterial reductases are fundamentally similar. It
may be taken as certain that the 2'-hydroxyl group is reduced
directly without the existence of intermediate products [Fig.
2(a)]. The experiments mentioned below rule out reaction
sequences such as dehydration-hydrogenation (b)or oxidationreduction (c), and activation of the hydroxyl group by phosphorylation (d) likewise cannot be detected, since synthetic
cytidine-2'-phosphate-S-diphosphateis not a reductase substrate" ']. In both systems, only one new hydrogen atom enters
the molecule with retention of configuration at the asymmetric
C2'.This follows from ~ h e m i c a 1 "and
~ ~NMR-spectroscopic
a n a l y ~ e s of~ the
~ ~ monodeuterated
. ~ ~ ~
(or monotritiated) 2'deoxyribonucleotides obtained when the enzymatic reduction
is carried out in 'HzO or 3 H z 0 and hence with deuterated
or tritiated dithiols R(S*H)zas the reducing agent. The assignment of the enzymatically formed prQducts has also been
confirmed by independent synthesis of the two epimeric [2'-(aor J3-)deuter0]-2'-deoxycytidines'~~~.
These results are compatible only with the direct pathway (a), and at the same time
refute an S Nsubstitution
of the hydroxyl group with inversion,
which is improbable in any case at the furanose ring.
The coenzyme B, 2-dependent enzyme diol dehydrase from
Aerohacter uciogmc~scatalyzes an oxidoreduction of propanediol to propionaldehyde, which seems to be related to the
reduction of ribonucleotides, and which proceeds with migration of oxygen cia an epoxide intermediate [reaction (4)If"'.
By reaction of specifically '0-labeled adenosine triphosp h a t e ~ [ ' ~with
l Lactobucillus ribonucleotide reductase, we have
HO-C-C -C H3
- "o/
shown that no intramolecular migration of the oxygen atoms
2' and 3' occurs in this system; 02'disappears completely
from the molecule, and 03'in [3'-180]ATP is not affected
by the reduction. However, if [2'-IsO]ATP is incubated with
enzyme and coenzyme in the absence of reducing agent, no
equilibration ofthe isotope content o f 0 2 'with Hz'"O occurs,
i.e. a conventional SN1 reaction with ionization at C 2 ' also
does not take place by route (a)['']. The isotope pattern of
the RNA and DNA from E. coli in a medium containing
'*O, which is identical apart from Oz'['al,shows that here
again the 2'-oxygen is eliminated by reduction in the same
direct way.
Nucleotides in which the free 2'-OH group is replaced by
a phosphate residue[711,by a methoxyf8' I , amino, azido, or
chloro substituent["I, or by an OH group in the arubino
configuration" 'lare not transformed by ribonucleotide reductase; however, 3'-xylonucleotides, 3'-deoxyribonucleoand 3'-0-methylribonucleotides are also not
reducedf8'! Since most of these compounds are not effective
inhibitors, we assume that the 3'-hydroxyl group is a binding
site for the enzyme, and that substrate binding (on 3'-OH)
and substrate reduction (on 2'-OH) are concerted. All these
findings can be formally compared to an S,i reaction, in
which the exchange of the hydroxyl group takes place in
a prefixed complex with several reaction partners (enzyme
groups and reducing agent) with retention of configuration.
There is much less clarity about the hydrogen transfer between
thioredoxin or another dithiol and the 2'-position of ribose.
There is growing experimental evidence that free-radical inter'mediates, which are stabilized by the cofactors (iron atoms
in E. coli, 5'-deoxyadenosylcobalamin in L. Ieichmunnii), occur
in this process. Thus an organic free radical that is characterized by a light absorption at 410nm and an ESR signal
at y= 2.0047, and whose destruction ( e .g. by hydroxylamine)
is exactly paralleled by a loss of the enzyme activity, is observed
in the metalloprotein B2ch7].A hydrogen-transferring function
of the coenzyme in the Lactobucillus reductase system can
be detected even more directly. It has been known for
some time that this enzyme, in the presence of dithiols and
an effector such as dGTP, catalyzes a tritium exchange between
synthetic [5'-3H]-5'-deoxyadenosylcobalamin and water
(which is used as an activity test) or conversely between 3 H Z 0
and unlabeled coenzyme, and that this tritium exchange and
the substrate reduction are inhibited to the same extent by
protein reagents such as N-bromosuccinimide[26.571.The reactive intermediate, in which there must be three equivalent
hydrogen atoms on C5' of the coenzyme, was recently discovered by fast-reaction technique^'^^.^^^; with a half-life in
the region of 10--100ms, one observes a change from the
spectrum of the coenzyme (with trivalent cobalt as a central
atom) to a spectrum of reduced cob(1i)alamin and the appearance of an ESR signal at g= 2.11 9, which can be reconciled
with an enzyme-fixed complex of the free radicals [)Co2'<]
and [5'-deoxyadenosyl.]. The concentration of this intermediate rapidly decreases again in the presence of substrate.
5'-Deoxyadenosine and aquocob( 1l)alarnin are also formed
in a slow, irreversible reaction, with the appearance of other
ESR signals[861.It may be assumed that the deoxyadenosyl
radical formed by reversible homolysis of the Co-C bond
in the coenzyme is responsible, in conjunction with one or
more thiol groups, both for the hydrogen exchange and for
the reduction of the ribose (Fig. 3).
1 B
5'- deoxyadenosylcobalamin
H- C-H
5 ' - deoxyadenosine
cob( i1)aIamin
Fig. 3 Structure and function of 5'-deoxyadenosylcobalamin (coenzyme B , I )
in the ribonucleotide reductase from L leichmannii (modified after [84]). The
transfer of a hydrogen isotope (*) from labeled coenzyme to water or from
water to the coenzyme or the substrate is shown. A=adenosine-C', B = p u rine or pyrimidine base, P = phosphate residues, R =thioredoxin or dithiol.
The c o r m ring system and the lower axial ligand of the cobalt are only
A separate analysis of the nature of the hydrogen transfer
as the final step of the reduction of substrate in such enzymeradical-thiol complexes will prove very difficult. No ESR signal
that could be assigned to a nucleotide radical has been
observed so far. It is therefore impossible at present to say
Angrw. Chem. incrrnat Edit.
Val. 13 (1974)
1 No. 9
whether the hydroxyl group is finally replaced by a hydrogen
atom or by a hydride ion. An answer to this question would
also require a precise knowledge of the amino acid side chains
in the catalytic center of the proteins, since the hydroxyl
group could, e. g., be specifically protonated by these side
chains and removed as a water molecule. (So far it has only
been shown that cysteine residues are probably involved in
the activity of both bacterial reductasesl“. *”.) From all the
observations taken together, one may expect a highly ordered
transition state from the substrate, amino acid groups of the
enzyme, and a reduced species from cofactors and SH groups;
Figure 4 is an attempt to describe this transition state in
the form of a “snapshot”.
fied nucleotides18’ - H J J , accelerate or inhibit the catalysis by
the enzyme, both the K , values and the rate constants of
the substrates being altered. Table 3 shows that there are
similarities in this respect among the type I enzymes, but
there are also great species-specific differences. A different
picture is found for the regulation pattern in the case of
the Lactohaiilltrs reductase (Table 4), where in addition to
slight product inhibition, a pronounced specific stimulation
of the reduction of ATP by dGTP, of CTP by dATP, and
of UTP by dCTP is observedfxyl:even totally non-natural
and slow-reacting nucleotides such such as (N-ribofuranosylbenzimidazole)-5’-triphosphate are subject to the stimulation‘”].
Fig. 4. Hypothetical model of the active site of a ribonucleotide reductase. The substrate (cytidine diphosphate) is fixed to the protein uia the terminal phosphate residues and the 3’-hydroxyl group; the
quality of the bonding to the purine or pyrimidine base depends on an ailosteric effector@. The reduction
of C2’is stimulated by other effectors @
,I which optimize the bonding of the hydrogen-donating system.
X is the iron complex or cob(ir)alamin radical of the type I and type I1 reductases respectively. A r r o
on the protein symbolize interactions of unknown nature. B=basic ami acid residue, P=phosphate,
R.R’= H o r OH.
In reality, however, the dynamic situation is even more complicated by the bonding of allosteric effectors to the enzyme.
3.2. Regulation of Enzyme Activity
Regulation of substrate reduction by effectors or modifiers
is so characteristic of ribonucleotide reductases that its description would fill many pages[xO.
5 7 . 6 2 . h 5 . 8 1 - 83.88 - 9 11. p rimarily,
deoxyribonucleoside triphosphates and ATP, and also modi-
Little is known about the detailed mode of action of the
effectors, as in most cases of this type. A number ofobservations
point to changes in the conformation of the protein, as is
required by the model of an allosteric regulation[3o.4 2 . h 5 . h61.
It is conceivable that the catalytic site o f a reductase is thereby
particularly well or less well ordered (Fig. 4). Extensive binding
studies[2’.6s’show that there are two classes of regulatory
siteson the E. coli subunits, one of which controls the substrate
specificity and the other the total activity of the enzyme.
Table 3. I’ffccl of nucleosidc triphnsphates on the reduction of rihonucleotidcs: \timulation I
I - ffcctor
I:. cofr [ 6 5 , 901
. .
inhih~tioii( - 1
Novikoff t u m o r [XX]
T4 phage [62]
+ ) and
. .
T.ihlc 4. Rclativc rcaction ratcs of thc redtiction of ribonuc1cotidt.s by Lu(rohu~i i l i i s reductase [ X I , XY].
C; T I’
ii ra AT I’
. .~
BzTI’ [b]
. ~ - ~ ...
~ .-~
[TI’ [a]
. . . . . . . . .
[a] lnosinc triphosphate.
[b] (N-Ribofiirant)sylbcnrlmldarolc)-~’-triphosphatc.
Anyew. Chrm. intrrnor. Edit.
1 Vol. 13 1 1 9 7 4 ) / No. 9
The Lactobacillus reductase also reacts to two groups of effector nucleotides, one of which again affects the substrate specificity and the other the enzyme-coenzyme interaction. including the tritium exchange'"! A regulatory effect of the coenzyme
concentration on the binding of substrate and effectors is
also observed'8y1. From the marked base specificity of all
these effects, considerable binding contributions by the amino
and keto substituents of the purine and pyrimidine bases
can be expected in the catalytic and regulatory centers: these
interactions have not yet been analyzed.
4. The Hydrogen-Donating Thioredoxin System
The hydrogen required for the reduction of ribonucleotides
ultimately comes from the cellular NADPH pool that is available for synthetic purposes, but it is used only through the
intervention of a specific "redoxin". As was mentioned earlier
(Section 2. l), the thioredoxin system,which was first discovered
in E. coli, consists of the small protein thioredoxin, whose
cysteine residues change reversibly between the dithiol and
disulfide forms, and the flavoenzyme thioredoxin reductase,
which is responsible for the reduction of the oxidized thioredoxin. If one includes the (probable) processes on enzymes
and coenzymes, one thus obtains a multi-stage hydrogen and
electron transport from NADPH to ribose (Fig. 5).
From the regulation phenomena listed in Tables 3 and 4. it
seems natural to construct a homeostatic mechanism that
guarantees a balanced supply of all four deoxyribonucleotides to the cell. If one nucleotide accumulates during DNA
FAD( ox)
E' = -0.31 V
thioredoxin reduct ase
-0.25 V
F A D (r e d )
e n z y m e fixed
thioredoxin ( r e d )
(? )
r i b o n u c l e o t ide r e duct a s e
Fig. 5 Intermediate steps of the hydrogen transfer from NADPH lo ribonucleotides by thioredoxin reductase and ribonucleotidc
reductase. In the penultimate stage. the structure of the 5'-deoxyadenosyl residue (type 11 reductases) is indicated (cf, Fig 3). P = phosphate residues, B = purine or pyrimidine base.
synthesis, it stimulates production of the others; on the other
hand, if no further DNA is being formed, an accumulation
of certain nucleotides inhibits further reduction of ribonucleotides. Detailed models of this kind have been devised[**-9'1.
The extent to which this regulation is actually achieved in riro
is admittedly difficult to estimate from the behavior of the
isolated ribonucleotide reductases at partly unphysiological
concentrations. Further unknowns are the rate and control
of the biosynthetic pathways by which effector nucleotides
(particularly dTTP) must first be formed from the primary
reduction products (e.g. dUDP) before they can exert a feedback control[t31.However, changes in the deoxyribonucleotide pools in tissue cultures under the influence of thymidine
and other nucleosides in the medium point to a functioning
of ailosteric regulation in intact cells'' "1.
Thioredoxins are heat-stable polypeptides built up from about
100 amino acids and having a molecular weight of about
12000, which have been isolated from the phage T4, from
bacteria, from yeast, and from liver (Table 5). They themselves
have no enzyme activity, but as an active site they contain
two cysteine residues separated by two other amino acids:
the sequence of this region in E. coli and yeastf'01"021, is
-Trp-Cys-Gly-Pro-Cys-Lys-, while the sequence in the T4
phage is -Lys-Cy~-Val-Tyr-Cys-Asp-[~~!
Oxidation to the disulfide with formation of a 14-membered ring is accompanied
by considerable local changes in conformation, which are
directly reflected in the changing tryptophan fluorescence of
the molecule^^'^^^. Like glutathione reductase. the NADPHspecific thioredoxin reductases, whose existence has also been
detected in several tissues (Table 5). contain FAD as the
Table 5. Molecular w i g h t s and other properties of thioredoxin and thiorcdoxin rcdiictascs.
. ~ . .~
. . ~ .
E x hrri(.hru coli
T4 phage
Lul.rohuc illirs Ilk
Rat liver
Calf liver
Novikoff tumor
. ~ . .
. . . . ... .
. . .~~.
I 1637. IOX amino acids. 2 ('ya
X7 amino acids. 2 C'ys
12600, ( ' L I . 120 amino acids. 4 C')s
not yct purified t o homogencity
12000. 103 amino acids. 4 C-ys
I 2 100, 109 amino acids, 6 Cys
. ~ .~.
~.. .
Thiorcdoxin i-cductasc
. . ~.
. ~
~ . .~ ..
. ..
. .
.. ~ . .
. ~ . . .
66000---73000. 2 prptide chains. 2 FAD
. .
[92.99. 101, 1031
~ 4 1
75000, 2 subunits. 2 FAD
66 700
~ 7 1
102. I I Y ]
[Yh. 1001
Angrw. Chem. Inrernar. Edir. J Vol. 13 (1974) f No. 9
prosthetic group; a disulfide grouping with the sequence
-Ala-Cys-Ala-Thr-Cys-Asp- also takes part in the redox pro-
cess in the E . cofi
Thioredoxins, thioredoxin rcductases, and ribonucleotide
reductases of different species have varying activities in heterologous mixtures, and no systematic pattern of interchangeability has been recognized so far. The readily obtainable E.
cofi thioredoxin has proved suitable as a hydrogen donor
for the Lwfohuc~i11rr.sand yeast ribonucleotide reductases"05.321, but is inactive toward the reductases of the T4
phage and of
Whereas the E. cofi ribonucleotide
reductase accepts all known thioredoxins, the thioredoxin
reductase of the same bacterium is strictly specific for its
own oxidized thioredoxin and that of T4 p h a g e ~ ' 3 ~ y ' ~ y x 1 .
Reductions of ribonucleotides and of thioredoxin show practically no connection with other metabolic pathways in the
cell. Only the participation of the thioredoxin system in the
reduction of methionine sulfoxide and of sulfate in yeast has
been detected" Oh], and the nitrate reduction and the ribonucleotide reduction in anaerobic microorganisms could have
a common hydrogen-donor system'"! The ribonucleotide
reduction, in accordance with Figure 5, is not reversible:
from what was said earlier, a general biosynthesis pathway
from deoxyribonucleotides to ribonucleotides is not to be
expected. However, an enzymatic hydroxylation of thymidine
or 2'-deoxyuridine to ribothymidine or uridine occurs as the
first step in the oxidative conversion of thymidine into uraciliro'l. A pyrimidine-2'-deoxyribonucleoside2'-hydroxylase
isolated from the mold Ntwrosporu crussa catalyzes this reaction with oxygen, ketoglutarate, iron( 11) ions, and ascorbate
as substrates and cofactors.
5. Ribonucleotide Reduction and Growth
Ribonucleotide reductase cannot be detected in dry wheat
grains or in unfertilized sea urchin eggs, but within a few
hours after the start of germination or after fertilization,
extracts of these tissuesexhibit theability to reduce ribonucleotides133.35! If part of the liver is removed from rats, the
ribonucleotide reductase activity of the regenerating tissue
increases to 20 times the normal value between 20 and 50 h
after the operation'"'. 431, and the deoxyribonucleotide pools
show a pronounced increase at the same time[10X1.In the
brains of chick and rat embryos, a pronounced maximum
of this enzyme activity roughly coincides in time with the
fastest proliferation of neur~blasts["~!
The key function of the ribonucleotide reductase for cell growth
becomes even clearer when its activity (or the size of the
dNTP pools) is followed during the division cycle and is
compared with the DNA synthesis in cultures of synchronized
cells. In mouse L cells(41Jand (particularly clearly, see Fig.
6) in yeast cellsr10Y1,
the ribonucleotide reductase reaches its
peak shortly before the strongest DNA production during
theS phase(i. e. the synthesis phase between two cell divisions).
The quantity of the four deoxyribonucleotides present in
synchronized mammalian cells (mouse, hamster)" 91 also
increases by a factor of 10 only during the S phase and
decreases in the early G 2 phase, during or shortly after the
DNA synthesis reaches its maximum.
Angrw. Chrm. internal. Edit. / Vo). I3 ( 1 9 7 4 )
1 No. 9
From the action of inhibitors of mRNA and protein biosynthesis such as a c t i n ~ r n y c i n [ ~puromycin[3s',
or cycloheximide[41.1091 In
. many of these tissues, it is quite clear that
the increase and decrease in the ribonucleotide reduction is
in fact due to induction or absence of t k - n o w enzyme synthesis.
The early appearance of ribonucleotide reductases in growing
cells can be easily reconciled with the stepwise onset of the
production of mRNA, protein, and DNA1'l o l . On the other
hand, no pronounced growth-dependent variations of this
kind have been observed for preceding and succeeding enzymes
such as thioredoxin reductasellO"i and DNA polymerase[3 s . 4 1 I
008 -
0 05
Fig 6 Ribonucleotidc reductasc activity ( 0 ) and DNA production 1 D
A ) in synchronized yeast cells (after [IOS]). Left-hand ordinate: nmol of
substrate rsactcd per mg of protein per min. right-hand ordinate: mg of
DNA per lOOml of ctiltiirc. abscissa: incubalion lime of the yeast cidlurc.
Ribonucleotide reductase is just as essential for the bacterium
Eschrrichiu cofi as for eukaryotic cells. Two mutants E 101
and LD195[1121
in which the proteins B1 and B2 of the reductase exhibit only a small fraction of the activity of the wild
type and are temperature-sensitive have recently been isolated.
The gene locus drzaF (one of seven that are responsible for
DNA synthesis) was thus identified as the structure gene for
ribonucleotide reductase. These bacteria can multiply at 37 to
42 C only if deoxyribonucleosides are supplied in the medium,
and growth inhibition by h y d r o x y ~ r e a [is~ *20~times as strong
as in the case of the wild type. Similarly, coenzyme B I may
be limiting for the type I1 reductases. The nodule bacterium
Rhizohiirm melilofi exhibits growth disturbances and a reduced
DNA content in the cells if it lacks cobalt, the central metal
atom of deoxyaden~sylcobalamin~~
Their significant involvement in the DNA synthesis and in
cell proliferation makes the ribonucleotide reductases (and
their inhibition) a subject of great current interest in virusinfected tissues and in tumor cells. T4 phages contain three
genes nrdA, B, and C;. phage-specific ribonucleotide reductase
and thioredoxin (but not thioredoxin reductase) that have
only a remote similarity to the bacterial proteins are formed
in the host cell as the products of these gene^[^^.^^.^^.^^^!
Increased specific reductase activities are found in kidney
cells infected with polyoma
in Yaba monkey virus
t ~ i m o r s ~and
~ ~ ”in, leukemic mice1461,but there are generally
no qualitative differences in relation to normal mammalian
enzymes. Until pure enzymes from normal and from infected
tissues are available, it will therefore not be possible to say
for certain whether pathogenic plant and animal viruses, and
in particular the oncogenic RNA viruses, have specific ribonucleotide reductases. N o obvious differences in the reductase
systems have been found in rat tumors and healthy rat h e r s .
Nevertheless, the reduction of ribonucleotides in tumors merits
great attention from the diagnostic and chemotherapeutic
standpoints, since E/ has been able to demonstrate a striking correlation between the rate of growth of numerotis rat
hepatomas and this e n z y m e a ~ t i v i t yThe
~ ~ ~very
~ : rapidly proliferating Novikoffand Walker tumors exhibit reductase activities up to 500 times as high as those of the slowest-growing
tumors. Relationships are conceivable between the cytotoxic
action of hydroxyurea and arabinonucleosides for malignant
and normal tissues in rir,o and their inhibitory effect on
ribonucleotide reductases in citro‘”. ’*.’” I s ] . . b0th Substances most strongly inhibit DNA synthesis and have less
effect on the RNA and protein syntheses. On the other hand,
it must not be forgotten that the nucleic acid metabolism
of a cell involves two dozen nucleotides and not many fewer
enzymes, so that the role of an individual enzyme, though
important, can hardly be the sole determining factor in a
complex process like DNA replication.
6. Outlook
unci to nwneroiis other colleaguesjor their kindness in communicating new re.sLc1r.T.
Received. January 18, 1974 [A 13 IE]
G e r m a n version: Angew. C h e m 86.624 (1974)
Translated by Expreas Translation Service. London
Hiirimiiii a n d S. K i i in D. M. G r c ~ h c ~ Meidbolic
Academic Press. New York 1970, 3rd Edit.. Vol. 4, Chaps. 19 and 20.
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Numerous chemically and biologically interesting aspects of
the reduction of ribonucleotides require further elucidation:
reaction mechanism, structure of the active sites and mode
of action of the effector molecules on the protein, initiation
and repression of enzyme biosynthesis. Though the initiating
factors of the enzyme induction are still completely unknown,
regulatory intervention in normal and in pathological .cell
growth by means of these key enzymes should be feasible
in the future. The existence of two entirely different ribonucleotide reductase systems for the catalysis of a fundamental metabolic reaction is remarkable. We feel that a comprehensive
characterization of the ribonucleotide reduction and the thioredoxin system in other organismsat different levelsofevolution
is of the greatest importance in order to clarify the distribution
of the enzyme types and regulation patterns. This may be
expected to yield new information about the evolution and
phylogenesis of life, since most models of the origin of life
take no account of the transition from ribonucleotides or
R N A to DNA as self-reproducing material, a step that is
scarcely imaginable without enzymes” l6]. Finally, the search
for new, possibly more easily handled ribonucleotide reduct a m is of interest from the standpoint of preparative nucleotide
chemistry, since it could facilitate access to modified deoxyribonucleotides from the much more readily accessible ribonucleotides. The recent rapidly growing interest in the reduction
of ribonucleotides should provide an answer to some of these
Our own work in this.field has been supported b y the Deiitsche
Forschirngsyumeinschaft. 1 urn also indebted to Harrj. Hogenkonzp anti Ray B l a k i q (Iowa City) .for their generous support,
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enzymatic, reduction, deoxyribonucleotides, ribonucleotide, pathways, biosynthesis
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