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INTRODUCTION |
Ribonucleotide reductase
(RNR),1 which catalyzes the
first reaction committed to DNA synthesis, is distinctive in many ways. First, the interplay between two different allosteric sites evidently acts to ensure that the four deoxyribonucleotide products of the enzyme
are synthesized at rates proportional to the base composition of the
genome of the organism (1). Because deoxyribonucleotides have no known
functions in eukaryotic cells except as DNA precursors, it makes good
metabolic sense for rates of synthesis and utilization to be balanced.
Second and less thoroughly investigated, the four ribonucleotide
substrates are reduced at one active site. To what extent does
competition among the four ribonucleotide substrates for binding to
this site contribute toward the ability of the enzyme to act in concert
with the need of the cell for dNTPs? It was partly for this reason that
our laboratory developed an assay protocol that allows all four RNR
reactions to be monitored simultaneously, in one reaction mixture (2,
3). Our results, obtained with ribonucleotide reductases encoded by
bacteriophage T4 (2) and vaccinia virus (3) support the conclusion that
ribonucleotide substrate concentrations are equal in importance to
concentrations of nucleoside triphosphate effectors as determinants of
the ability of the enzyme to produce its four products at rates
proportional to the base composition of the genome.
We have used the vaccinia virus enzyme as a model for mammalian
ribonucleoside diphosphate reductases, because of its close structural
relationship to the mammalian enzymes and because the viral enzyme
subunits have been available in our laboratory for some time as
purified recombinant proteins (4). In applying the four-substrate assay
to vaccinia virus RNR, we observed one striking phenomenon: a fairly
strong and quite specific inhibition of the reduction of ADP by ADP
itself (3). We speculated that this effect might represent a metabolic
control system that links the energy status of a cell with systems for
DNA replication. Before carrying out experiments to test this
hypothesis, it was essential to learn whether this effect is peculiar
to the viral model system we were using or whether a mammalian
ribonucleotide reductase behaves similarly. Within the past few years,
cDNAs for the large (R1) and small (R2) subunits of the mouse RNR
have become available (5, 6). Therefore, using recombinant mouse ribonucleotide reductase, we have now been able to ask whether ADP
behaves similarly as an apparent regulator of enzyme activity. It was
also of interest to confirm that the effects of allosteric modifiers,
seen in vitro in analysis of single-substrate reactions, are
similar when the enzyme is acting upon all four substrates simultaneously. Finally, we wished to extend our earlier observation (3) that interspecific hybrid RNRs, formed by mixing mouse and vaccinia
virus subunits, are enzymatically active.
Like all known mammalian RNRs, the enzymes encoded by phage T4 and
vaccinia virus are type I ribonucleotide reductases (1), which act upon
ribonucleoside diphosphate (rNDP) substrates. Each enzyme is a
heterotetramer containing the homodimeric R1 and R2 proteins. The R2
protein contains a catalytically essential tyrosine free radical formed
and stabilized with the help of a nearby iron center consisting of two
ferric ions bridged by an oxygen atom. The R1 protein contains the
catalytic site and two allosteric sites: the activity site, which binds
either ATP or dATP and regulates overall catalytic activity, and the
specificity site, which binds either ATP, dATP, dGTP, or dTTP and
regulates specificity; for example, dTTP, when bound in the specificity
site, activates the reduction of GDP and inhibits the reduction of both
CDP and UDP.
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EXPERIMENTAL PROCEDURES |
Expression of Mouse cDNAs for R1 and R2 and Purification of
the Recombinant Proteins--
Mouse R1 and R2 cDNAs were expressed
in Escherichia coli BL21(DE3), from plasmids developed in
Lars Thelander's laboratory (Umeå, Sweden) (5, 6) and kindly provided
by Dr. Thelander. Induction and purification of mouse R1 protein were
carried out as described (6), except that bacteria after harvesting
were lysed in a French pressure cell at 15,000 p.s.i. Also, elution of
the protein from a dATP-Sepharose affinity column was carried out with
column buffer containing 75 mM ATP, a condition that we had
found earlier to be effective in purifying vaccinia virus R1 (4). Mouse
R2 protein was induced and purified as described by Mann et
al. (5) and as modified in our laboratory (3). Recombinant
vaccinia virus R1 and R2 proteins were expressed and purified as
described previously by this laboratory (4, 7).
Four-substrate Assay for Ribonucleotide Reductase--
Enzyme
assays were carried out essentially as described previously (2, 3),
with modifications as indicated in individual tables and figure
legends. Reaction mixtures contained 50 mM HEPES, pH 8.2, 5 mM MgCl2, 50 mM dithiothreitol, 20 µM
Fe(NH4)2(SO4)2, 1 µM mouse R1 protein, 4 µM R2 protein, and
ribonucleoside diphosphate substrates and nucleoside triphosphate
effectors at the indicated concentrations and in a total volume of 100 µl. Incubation was carried out at 37 °C for 5 min. Reactions were
terminated by addition of 5 µl of 50% (v/v) perchloric acid, and
this was followed by chromatographic analysis of the reaction mixture
(2, 3).
The pH value used for the assay reactions, 8.2, was based upon our
earlier determination of the pH optima for RNRs of vaccinia virus and
cultured BSC40 monkey kidney cells (8). When we ran the four-substrate
assay with the mouse enzyme at pH 7.6 under our quasi-physiological
conditions, we found the activity to be decreased by about 24%
compared with the activity determined at pH 8.2 and the overall product
profile to be substantially the same (data not shown).
Nucleotide Binding Experiments--
Nucleotide binding to mouse
R1 protein was measured according to the ultrafiltration assay devised
by Ormö and Sjöberg (9). For each assay a
3H-labeled dNTP, with or without added unlabeled rNDPs, as
indicated, was mixed with R1 in a solution containing 50 mM
Tris-HCl, pH 7.6, 10 mM MgCl2, and 2 mM dithiothreitol in a total volume of 150 µl. After
incubation on ice for 15 min, a 30-µl aliquot was taken for
scintillation counting to quantitate total nucleotide concentration.
The remainder (120 µl) was applied to a Nanosep centrifugal separator
(Gelman; cut-off value, 30 kDa), and centrifugation at 6000 rpm was
carried out for 2 min. Aliquots of 30 µl were then withdrawn from the
filtrate for scintillation counting to quantitate unbound nucleotide.
Concentrations of bound nucleotides were determined by subtracting
unbound nucleotide from total nucleotide. Control reactions run in the
absence of added protein showed that no significant amounts of
nucleotide were bound nonspecifically to the filters; in these control
reactions the counts in "unbound" nucleotide were virtually
identical to counts in "total" nucleotide.
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RESULTS |
Regulatory Effects of Individual Nucleoside Triphosphate
Effectors--
With the exception of our preliminary studies on T4 and
vaccinia virus ribonucleotide reductases, most of what we know about allosteric control of this crucially important enzyme has been learned
from assay protocols in which the enzyme is exposed to only one
substrate at a time. Within the cell, the enzyme is exposed to all four
rNDP substrates. How might competition among these substrates influence
the activities of individual allosteric effectors?
Fig. 1 explores the effects of individual
allosteric modifiers in the four-substrate assay, as well as
illustrating the kind of data generated by this assay. As noted earlier
(2, 3), we remove unreacted rNDP substrates and ATP from the reaction mixture by boronate column chromatography, and then the
deoxyribonucleoside diphosphate products of the reactions are resolved
by anion exchange high pressure liquid chromatography. These profiles
also include the individual dNTP effectors added, but not ATP, which is
removed by the boronate column. In this experiment the four rNDP
substrates were present initially at equal concentrations of 0.15 mM each. ATP was present at 2.5 mM, its
approximate intracellular concentration, whereas each individual dNTP
was present at 40 µM. At this level, dATP was expected to
bind primarily to the specificity site and less significantly to the
activity site.

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Fig. 1.
Regulation of mouse ribonucleotide reductase
activities in the four-substrate assay by individual allosteric
effectors. All reaction mixtures contained the four rNDP
substrates at 0.15 mM each and ATP at 2.5 mM.
Individual dNTP effectors, where indicated, were added at 40 µM each.
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As shown by Eriksson et al. (10) for the calf thymus RNR, we
found the mouse enzyme to be completely inactive in the absence of any
added nucleoside triphosphate (data not shown). The data in Fig. 1 show
that individual dNTPs have effects similar but not identical to those
described for calf thymus (10) and mouse (11) RNRs in single-substrate
assays. dCTP had no significant effect on any of the four activities
when results with ATP plus dCTP were compared with results seen when
ATP was the only effector added. dTTP strongly activated reduction of
GDP and ADP, while inhibiting CDP reduction. dGTP greatly stimulated
ADP and GDP reduction, while inhibiting the reduction of both
pyrimidine nucleotides. dTTP activated GDP reduction much more strongly
than ADP reduction, whereas dGTP had the converse effect. The
activation of ADP reduction by dTTP and the activation of GDP reduction
by dGTP are effects not seen in single-substrate analysis of calf
thymus RNR (10) or recombinant mouse RNR (11).
dATP slightly activated ADP reduction and strongly inhibited CDP and
UDP reduction, whereas GDP reduction was undetectable. This suggests
that dATP added at 40 µM does bind significantly to the
activity site. In additional experiments (not shown), dATP was seen to
inhibit all four activities at higher concentrations, as has been seen
repeatedly in single-substrate assays. Numerical values are presented
in Table I. These data show the mouse
enzyme to be significantly more sensitive to individual allosteric
effectors than vaccinia virus RNR, assayed under similar conditions
(compare with Fig. 2 in Ref. 3). For
example, the GDP reductase activity of the viral enzyme was
approximately doubled by dTTP and halved by dATP. By contrast, the
mouse GDP reductase activity was absolutely dependent upon added dTTP
and was completely inhibited by added dATP at the concentration used.
Similarly, the inhibitory effect of dGTP upon CDP and UDP reduction
appeared to be stronger for the mouse than for the viral enzyme.
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Table I
Effects of allosteric modifiers on activities of mouse
ribonucleotide reductase
Each reaction mixture contained ADP, CDP, GDP, and UDP at 0.15 mM each and ATP at 2.5 mM. Each of the four
activities was negligible when ATP was omitted from the reaction
mixture.
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Fig. 2.
Effects upon mouse rNDP reductase specificity
of proportional variations in rNDP substrate concentration. The
bioproportional substrate mixture contained ADP, CDP, GDP, and UDP at
12.0, 1.0, 2.0, and 2.0 mM, respectively. Allosteric
effectors were present at their estimated in vivo
concentrations (2.5 mM ATP, 60 µM dATP, 15 µM dGTP, and 50 µM dTTP).
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Enzyme Behavior in the Presence of Four rNDP Substrates at
Estimated in Vivo Concentrations--
We presume that ribonucleotide
reductase in vivo acts upon the four rNDP substrates at
relative rates corresponding to the nucleotide composition of the
genome. Mouse DNA contains 58% A + T and 42% G + C (12). Table
II presents enzyme product profiles for
reactions run under various conditions. For these experiments, quasi-physiological or in vivo concentrations of nucleoside
triphosphates were estimated from pool size determinations in our
laboratory for a variety of cultured mammalian cell lines (3), and
quasi-physiological concentrations of rNDP substrates were estimated
from nucleotide pool data compiled by Traut (13) for cultured mammalian
cell lines. Traut estimated the following values for intracellular rNDP
concentrations from averaged data from many studies of nonhuman cell
lines: ADP, 849 µM; CDP, 71 µM; GDP, 159 µM; and UDP, 155 µM. We rounded off these
values as follows for our quasi-physiological conditions: ADP, 850 µM; CDP, 70 µM; GDP, 160 µM;
and UDP, 160 µM. Of course, these estimates could be
quite inaccurate because of variation among different cell lines,
compartmentation, variation of activity coefficients within the
intracellular milieu, and so forth. Nevertheless, our data clearly
establish the essentiality of allosteric modifiers for directing the
enzyme to produce four deoxyribonucleoside diphosphates at rates
corresponding to their rates of utilization.
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Table II
Effects of allosteric modifiers and substrate concentrations upon mouse
ribonucleotide reductase product profiles
All reaction mixtures contained ATP at 2.5 mM. Equimolar
substrates refers to 0.15 mM each of ADP, CDP, GDP, and
UDP. In vivo substrates refers to 850 µM ADP,
70 µM CDP, 160 µM GDP, and 160 µM UDP. ATP was present in each reaction mixture at 2.5 mM. In vivo dNTPs refers to 60 µM
dATP, 15 µM dGTP, and 50 µM dTTP.
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The importance of substrate concentrations as a determinant of
physiological specificity is evident but less pronounced than what was
seen earlier for the vaccinia virus enzyme (3). At equimolar rNDPs (150 µM each), ADP reduction was significantly higher and GDP
reduction was significantly lower than expected for biologically
balanced deoxyribonucleotide production. At quasi-physiological concentrations, ADP reduction was close to the expected value, and GDP
reduction was slightly closer to but still below the expected value.
This presents a contrast from comparable results with the vaccinia
virus RNR, in which we found that GDP reduction is higher than expected
under quasi-physiological conditions. The more striking contrast,
however, is for UDP reduction, where we found the vaccinia enzyme to
have little activity under all conditions tested. Table II, however,
shows the relative rate of UDP reduction by the mouse enzyme to
correspond fairly closely to the representation of dTMP in the mouse
genome. Others have reported (14) that most of the dTMP in mammalian
genomes arises from dCMP, via the dCMP deaminase reaction
followed by thymidylate synthase and that intracellular reduction of
UDP to dUDP is negligible under most conditions. The data of Table II
show CDP reduction at quasi-physiological rNDP concentrations to be
considerably higher than the proportion of dCMP in the mouse genome.
Therefore, even though UDP reduction is significant, our data are
consistent with the concept that a significant fraction of the dTMP
residues in mouse DNA arises via deamination of
deoxycytidine nucleotides.
Effects of a Range of Substrate Concentrations--
Although our
estimates of effective intracellular rNDP concentrations may be
inaccurate for the above-mentioned reasons, we felt that the rNDP
concentrations relative to each other should more closely reflect the
in vivo situation. Therefore, we carried out a substrate
concentration-velocity experiment, using a bioproportional mixture of
ribonucleoside diphosphates as the variable substrate rather than a
single substrate. According to the mammalian rNDP pool data compiled by
Traut (13), the ratio of ADP to CDP to GDP to UDP is about 12:1:2:2.
Fig. 2 shows results of an experiment in which ADP concentration was
varied from 300 to 1800 µM and the other three rNDPs were
varied proportionately. None of the concentrations tested yielded a
closer fit between the product profile in the four-substrate assay and
the nucleotide composition of mouse DNA. This result suggests either
that our assumptions about rNDP concentration ratios in mouse cells are
inaccurate, that control of rNDP reductase specificity in
vivo involves additional factors, or both.
The most interesting feature of Fig. 2 is the marked inhibition of
purine nucleotide reduction seen at the highest substrate concentrations. In this respect the mouse RNR resembles vaccinia virus
RNR (3), and this result suggests that the apparent control of RNR
activity by ribonucleoside diphosphates might be physiologically significant. To further investigate the basis for this effect, we
varied the concentration of each rNDP individually, in the presence of
each of the other three rNDPs at its respective estimated in
vivo concentration. As shown in Fig.
3, ADP inhibited its own reduction at
high concentrations, although the effect was not as pronounced as seen
in the experiments with bioproportional substrate mixtures. The effect
of ADP is evidently more complex than simple competition with other
rNDPs for catalytic sites on the enzyme, because CDP reduction was
activated at high ADP concentrations; if only competition at the
catalytic site site were involved, CDP reduction would decline at
higher ADP concentrations, and ADP reduction would increase. By
contrast, the effects of the other rNDPs did seem to involve mostly
competition for binding at the catalytic site. In Fig.
4, for example, note that increasing GDP
concentration in the presence of fixed amounts of the other three rNDPs
increased the rate of GDP reduction but decreased the activity of the
enzyme toward the other three substrates. A nearly identical result was
seen with CDP, namely, increased CDP reduction at high levels coupled
with decreased activities toward the other three substrates.

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Fig. 3.
Effects upon mouse rNDP reductase specificity
of variations in ADP concentration at constant concentrations of the
other three rNDPs. ADP concentrations were varied as shown,
whereas concentrations of the other three rNDPs and the allosteric
effectors were held constant at their estimated quasi-physiological
concentrations.
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Fig. 4.
Effects upon mouse rNDP reductase specificity
of variations in individual substrate concentrations. For each of
the three experiments shown, the concentration of one rNDP was varied
as indicated, whereas the levels of the other three rNDPs and the
allosteric effectors were held constant at their estimated in
vivo concentrations.
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Analysis of Nucleotide Binding to Mouse R1 Protein--
The enzyme
activity data presented above indicate that the effect of ADP on the
RNR product profile is more complex than simple competition between ADP
and other rNDP substrates at the catalytic site. Does ADP bind to a
previously unknown site on R1, or does it bind at high concentrations
to the specificity and/or activity sites? As an approach toward
answering that question, we carried out nucleotide binding experiments,
using the ultrafiltration assay of Ormö and Sjöberg (9).
The immediate goal of these experiments was to determine whether ADP,
either alone or in combination with other rNDPs, influences binding of
dNTP effectors to the high affinity specificity sites of R1. First it
was necessary to quantitate the binding of dNTPs to R1 in the absence
of added rNDP substrates. Data from these experiments are shown in Fig. 5. Both dGTP and dTTP displayed
hyperbolic binding behavior as expected for single-site binding. Curve
fitting for the dGTP data (Fig. 5A) yielded a
Kd of 4.4 ± 0.38 µM and for the
dTTP data (Fig. 5B) a Kd of 1.8 ± 0.30 µM. Because dATP binds at two sites with differing
affinities, we carried out dATP binding experiments at two different R1
protein concentrations and displayed the data on a Scatchard plot (Fig.
5C). Linear regression analysis yielded for the high
affinity (specificity) site a Kd of 12.6 ± 2.3 µM and for the low affinity (activity) site a
Kd of 54.3 ± 4.0 µM.

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Fig. 5.
Determination of binding constants of
allosteric effectors to mouse R1 protein. Binding was analyzed by
ultrafiltration (9). A, dGTP binding; B, dTTP
binding; C, dATP binding. For the experiment in
A, R1 protein was present in each binding reaction at 6 µM, and in B, R1 was present at 2 µM. Kd values were determined by
nonlinear least squares fit by using Kaleidagraph 3.5. Because dATP
binds at two sites, data were collected at two different R1
concentrations as shown on the figure, and data were analyzed by linear
regression on a Scatchard plot.
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Our first experiment tested the idea that ADP at high concentrations
can bind, as an ATP analog, either to the specificity site on R1, to
the activity site, or to both. Accordingly, we asked whether ADP at
high concentrations could inhibit the binding of dATP to R1. As shown
in Fig. 6, however, ADP at concentrations as high as 1800 µM had no discernible effect upon the
binding of dATP, present at 1.5 µM (open
circles). Because ADP in this experiment is expected to bind to
the catalytic site as well as to possible other sites, we modified the
protocol by asking whether ADP could inhibit dATP binding under
conditions where little if any ADP was bound at the catalytic site.
This was accomplished by running the binding assays in the presence of
CDP at a constant concentration of 320 µM, 10-fold higher
than the reported Km for CDP with a mammalian RNR
(11), so that ADP would be largely displaced from catalytic sites.
Under these conditions also (Fig. 6, closed circles) we saw
no effect of ADP upon amount of dATP bound. However, at all
concentrations of ADP, the presence of CDP had a slight but consistent
depressive effect upon the amount of dATP bound.

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Fig. 6.
Lack of effect of ADP upon dATP binding to
mouse R1 protein. Each binding reaction mixture contained 1.5 µM R1, 1.5 µM [3H]dATP, CDP
(where indicated) at 320 µM, and ADP at the indicated
concentrations.
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The latter finding suggested that CDP itself might have a direct effect
upon the binding of nucleotides at one or both allosteric sites. We
tested this idea by observing the effect of CDP upon the binding of its
prime allosteric inhibitor, dGTP. As shown in Fig.
7A (open circles), CDP at high
concentrations inhibited dGTP binding by nearly 10-fold. This finding
suggests that allosteric and catalytic sites are in two-way
communication. Most literature on allosteric enzymes focuses upon the
concept that effector binding at an allosteric site influences
substrate binding at the catalytic site. The data of Fig. 7A
indicate that the converse relationship also holds. The metabolic
rationale for this behavior of the enzyme is that CDP can promote its
own reduction by driving release from the enzyme of its prime
inhibitor. A similar relationship is shown in Fig. 7B
(open circles), where we show that GDP stimulates the binding of its allosteric activator, dTTP. In this case the data suggest that GDP promotes its own reduction by causing a shift in RNR
specificity toward GDP, through binding of an effector that is both
inhibitory to CDP and UDP reduction and stimulatory toward its own
reduction.

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Fig. 7.
Effects of ribonucleoside diphosphates on
binding of allosteric effectors to mouse R1 protein. A,
effect of CDP upon dGTP binding. Each binding reaction mixture
contained 6.0 µM R1, 6.0 µM
[3H]dGTP, ADP (where indicated) at 500 µM,
and CDP at the indicated concentration. B, effect of GDP
upon dTTP binding. Each binding reaction mixture contained 2.0 µM R1, 2.0 µM [3H]dTTP, ADP
(where indicated) at 500 µM, and GDP at the indicated
concentrations.
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Because the primary goal of these experiments was to understand the
effect of ADP upon ADP and GDP reduction, the nucleotide binding
experiments depicted in Fig. 7 were carried out in the presence of ADP
at a constant concentration. That concentration was set at 500 µM, a value permitting ADP to compete effectively with
CDP and GDP, respectively, for binding at the catalytic site. Again, as
in the experiment of Fig. 6, ADP displayed a small but consistent
effect. In Fig. 7A, note that ADP depressed the inhibition of dGTP binding by CDP, and in Fig. 7B, the effect of ADP
was to depress the stimulation of dTTP binding by GDP.
These experiments suggest a model for understanding the effects of ADP
upon activities of mouse RNR. As suggested by the data of Fig. 6, ADP
may be relatively inert in the sense that its binding to the catalytic
site does not directly influence affinities for effectors at the
specificity site. However, it might influence these affinities
indirectly, by competing with the other RNR substrates for binding to
the catalytic site and thereby depressing the effects of those other
rNDPs on effector binding. This idea was tested in the experiments of
Fig. 8, where ADP concentration was
varied in the presence of other rNDP substrates at constant
concentrations. As seen in Fig. 6, ADP by itself exerts little or no
direct influence on effector binding. Note from the squares
in both panels A and B of Fig. 8 that ADP had
little or no effect, in the absence of other rNDPs, on the binding of
either dGTP or dTTP. Note also confirmation of the results shown in
Fig. 7; in the absence of added ADP, CDP strongly inhibited dGTP
binding (compare circles with squares, Fig.
8A), and GDP activated dTTP binding (compare circles with squares, Fig. 8B). As ADP
concentration was increased, dGTP binding was increased in the presence
of CDP (circles, Fig. 8A), and dTTP binding in
the presence of GDP was decreased (circles, Fig.
8B). In Fig. 8A an even greater stimulation of
dGTP binding by ADP was seen when the experiment was carried out in the
presence of a mixture of CDP, UDP, and GDP, each at its estimated
physiological concentration (triangles). Taken together,
these data support the hypothesis upon which the experiment was based,
namely, that the effect of ADP binding to allosteric effectors is
indirect. From the experiment of Fig. 8A, we suggest that
ADP stimulates dGTP binding by displacing CDP from the catalytic site,
thereby limiting the inhibition of dGTP binding by CDP. Similarly, in Fig. 8B, we suggest that ADP binding displaces GDP, thereby
limiting the ability of GDP to stimulate dTTP binding.

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Fig. 8.
Indirect effects of ADP upon binding of
allosteric effectors to mouse R1 protein. A, dGTP
binding. All binding reaction mixtures contained 6.0 µM
R1 protein, 6.0 µM [3H]dGTP, and ADP at the
indicated concentrations. Squares, no further additions;
circles, CDP present in all mixtures at 320 µM; triangles, all mixtures contained 70 µM CDP, 160 µM UDP, and 160 µM GDP (quasi-physiological concentrations).
B, dTTP binding. All binding reaction mixtures contained 2.0 µM R1 protein, 2.0 µM
[3H]dTTP, and ADP at the indicated concentrations.
Squares, no further additions; circles, GDP
present in all mixtures at 500 µM; triangles,
all mixtures contained 70 µM CDP, 160 µM
UDP, and 160 µM GDP (quasi-physiological
concentrations).
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Activities of Mouse/Vaccinia Virus Chimeric rNDP
Reductases--
Although this study has documented some significant
differences between the ribonucleotide reductases of mouse cells and
vaccinia virus, the two enzymes are closely related, with amino acid
sequence identities near 80% (15). Partly to examine whether the close structural relationship connotes a close functional relationship, in
our earlier study (3), we showed that a hybrid enzyme containing vaccinia virus R1 protein and mouse R2 protein was enzymatically active, although the specific activity was lower than our values for
the native viral enzyme (4) and those determined by Davis et
al. (6) for the mouse enzyme. Because for the present study we had
prepared homogeneous recombinant mouse R1 protein, we repeated the
experiment, using both mouse/vaccinia hybrid enzymes. For this
experiment we assayed equimolar rNDP substrates (150 µM
each) in the presence of ATP as the sole allosteric effector,
conditions that maximize the reduction of CDP. As shown in Fig.
9, the vaccinia R1/mouse R2 hybrid is
active, as reported previously, with a specific activity significantly
lower than that of either native holoenzyme. Unexpectedly, the mouse
R1/vaccinia R2 hybrid was more active than either native enzyme, with a
specific activity about twice that of the vaccinia virus
holoenzyme.

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Fig. 9.
Activity of heterotetrameric rNDP
reductases. All assays were carried out with R1 present at 1 µM and R2 at 4 µM, with all four substrates
present at 0.15 mM each, and with ATP at 2.5 mM
being the only allosteric effector present. A, mouse
homotetramer; B, vaccinia virus homotetramer; C,
mouse R1 plus vaccinia virus R2; D, vaccinia virus R1 plus
mouse R2.
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DISCUSSION |
Simultaneous analysis of the four activities of ribonucleotide
reductase has revealed novel aspects of the regulation of this pivotal
enzyme, as shown both by our earlier studies of type I RNRs of T4 phage
and vaccinia virus (2, 3) and by more recent studies by Andersson
et al. (16) of the type III anaerobic RNR of T4 phage.
Recombinant mouse ribonucleotide reductase shows allosteric behavior in
the four-substrate assay similar to that earlier described for other
mammalian RNRs (10, 11), such as strong dependence upon dGTP for ADP
reduction, upon dTTP for GDP reduction, and upon ATP for all activities
of the enzyme. General inhibition by dATP and inhibition of CDP and UDP
reduction by dGTP are among the feedback inhibition phenomena described previously. Our studies reveal two regulatory effects, less strong but
still significant, which have not been reported before: activation of
ADP reduction by dTTP and of GDP reduction by dGTP. Whether these
effects play a significant role in regulating dNTP pool sizes in
vivo cannot yet be determined from our data.
The relatively high activity of the mouse enzyme toward UDP was
unexpected in light of the negligible activity of the vaccinia virus
enzyme toward this substrate, and in vivo data suggesting that the pathway dCMP
dUMP
dTMP 
dTTP is more significant in vivo for dTTP synthesis than is the pathway involving
RNR, viz. UDP
dUDP
dUMP
dTMP 
dTTP.
Perhaps there are additional factors that control UDP reductase
activity in vivo. In any case, the results presented here
suggest that it would be desirable to re-examine the roles of
ribonucleotide reductase and dCMP deaminase in dUMP synthesis in
vivo.
Regulation of mouse RNR activity by ADP seems to be less specific than
was reported previously (3) for the vaccinia virus enzyme, in the sense
that the bioproportional mixture of rNDPs exerts a stronger regulatory
influence under our conditions than does ADP alone. For example, in the
experiment of Fig. 2, the percentage of dADP + dGDP in the product mix
dropped from 64 to 28% in going from the lowest to the highest
rNDP concentration tested. By contrast, in the experiment of Fig. 3,
the corresponding change, in going from 800 to 1800 µM
ADP, was from 48% of the total to 34%.
Whatever the specific regulatory mechanisms involved, however, it is
apparent that ADP interacts with the enzyme differently from the other
rNDPs, because its effects upon the product profile in the
four-substrate assay cannot be explained simply in terms of competition
among the four substrates for the catalytic site. The nucleotide
binding experiments depicted in Figs. 6-8 suggest a model for
understanding this interaction. According to this model, ADP binds
exclusively at the catalytic site. Note from Fig. 6 that ADP at a
1200-fold molar excess over dATP had no detectable effect upon dATP
binding, suggesting that ADP does not interact with the two allosteric
sites. We cannot rule out the possibility of some binding of ADP at the
activity site, because this experiment was carried out at 1.5 µM dATP, a concentration so low that binding should have
occurred primarily at the high affinity specificity site. Nor can we
yet rule out the possibility that ADP binds to a previously undescribed
site on the R1 protein. More direct approaches are needed, and these
are in progress.
If we assume that the effects of ADP result from its interaction with
the catalytic site, we can understand its effects upon the specificity
of mouse ribonucleotide reductase, namely, activation of CDP reduction
and inhibition of ADP and GDP reduction. ADP evidently has little
direct effect on the binding of either dATP (Fig. 6), dGTP (Fig.
8A, squares), or dTTP (Fig. 8B,
squares), whereas CDP and GDP both have strong effects upon
binding of allosteric effectors (Fig. 7). However, ADP can modulate the
effects of CDP and GDP by competing with them for binding to the
catalytic site. The intracellular pool size of ADP, which is much
higher than those of the other three rNDPs (13), suggests that this
competitive binding is metabolically significant. A consequence of the
displacement of CDP and GDP from the catalytic site is to depress the
binding of dTTP (promoted by GDP) and increase binding of dGTP
(inhibited by CDP) at the specificity site; these events in turn should
inhibit reduction of ADP and GDP and increase reduction of CDP. Our
observation (Fig. 8A, triangles) that ADP shows a
3-fold stimulation of dGTP binding in the presence of
quasi-physiological concentrations of CDP, GDP, and UDP is consistent
with this interpretation.
The experiment of Fig. 5 yielded Kd values for the
binding of dATP, dGTP, and dTTP to mouse R1 protein. The values reported here (12.6 and 54.3 µM for dATP and 4.4 µM for dGTP) are considerably higher than corresponding
values recently reported (11) for mouse R1 (0.07 and 1.5 µM for dATP and 0.2 µM for dGTP). Although
we do not know the basis for the discrepancy, we note that the values
we have determined are closer to what one might expect a
priori if these interactions are to participate in metabolic regulation, because they lie closer to actual intracellular
concentrations of these effectors. The average intracellular
concentrations estimated by Traut (13) are 24 µM for dATP
and 5.2 µM for dGTP; these values lie close to those
determined in our own laboratory with several different cultured
mammalian cell lines (17).
Finally, the high activity of the chimeric RNR containing mouse R1 and
vaccinia virus R2 was unexpected; it would be of interest to determine
whether this higher activity results from particularly efficient iron
binding in vitro by the hybrid enzyme. However, our
observation may explain why early studies on targeted deletion of the
vaccinia virus R1 gene led to mutant viruses with no significant phenotype (18, 19). Perhaps a chimeric enzyme is formed in cells
infected by this mutant. In vaccinia virus-infected monkey kidney
cells, the R2 protein accumulates in significant excess over R1 (20).
The viral R2 present may suffice to drive the formation of a
significant amount of an enzymatically hybrid enzyme. Because levels of
cellular R1 vary with growth phase of a cell culture (21), a prediction
from our observations would be that the ability of a cell line to
support productive vaccinia virus infection would be a function of the
level of its own R1 protein.