|
|
|
|||||||
|
|||||||||
J Biol Chem, Vol. 273, Issue 45, 29512-29518, November 6, 1998
From the Department of Biochemistry and Biophysics, Oregon State
University, Corvallis, Oregon 97331-7305
As determined by simultaneous monitoring of its
four activities, vaccinia virus-coded ribonucleoside diphosphate (rNDP)
reductase shows responses to individual nucleoside triphosphate
effectors The allosteric regulation of ribonucleotide reductase
(RNR)1 by individual
nucleoside triphosphates is well characterized for several forms of the
enzyme (1, 2). The three classes of RNR, although quite different in
structure (3), have similar allosteric behavior in terms of the effects
of modifiers upon substrate specificity. In all classes, ATP and dATP
(when it is not inhibitory) activate reduction of the cytidine and
uridine nucleotides. Reduction of adenosine and guanosine nucleotides is stimulated by dGTP and dTTP, respectively. These effects are mediated through binding of nucleotides to two different allosteric sites on the dimeric R1 (large) protein: the activity sites, which bind
with relatively low affinity ATP (general activator) or dATP (general
inhibitor), and the specificity sites, which bind with higher affinity
ATP, dATP, dGTP, or dCTP, with the binding of each ligand activating
the inhibition of some substrates and inhibiting that of others.
However, due primarily to limitations of traditional RNR assay methods,
only the isolated effects of individual allosteric effectors have been
determined, in kinetic experiments involving single substrates. We have
developed an assay procedure (4) that permits simultaneous monitoring
of the four RNR activities. With this procedure, the effects of more
complex nucleotide environments on substrate turnover rates can be
examined. In addition, other factors that influence relative turnover
rates, such as protein-protein interactions, can be evaluated by this technique.
Because of the ease with which it can be purified in large quantities
as a recombinant enzyme, we are using the class I RNR encoded by
vaccinia virus as a model for the closely related mammalian cell
enzymes. The two viral RNR genes express proteins similar in size to
mammalian R1 (large) and R2 (small) RNR proteins, and the molecular
weight of the holoenzyme suggests the Overexpression and Purification of Recombinant
Subunits--
Both subunits of vaccinia ribonucleotide reductase were
previously cloned into
isopropyl-1-thio-
The purification procedure for the vaccinia R2 subunit was essentially
as reported (5), except for the incorporation of an additional radical
reactivation step, described in the accompanying paper (11). Mouse R2
protein was purified by the same procedure used to purify the vaccinia
R2 (5). Purification of the vaccinia R1 protein was performed also as
described in the accompanying paper (11).
Ribonucleotide Reductase Four-substrate Assay--
The assay was
carried out as described earlier (4). Essentially, the technique
involves two chromatographic steps for resolution of complex reaction
mixtures. The first step, boronate affinity chromatography, separates
the reaction products (dNDPs) from ATP and unreacted substrates
(rNDPs). In the second step, the dNDPs are resolved into individual
nucleotides and separated from dNTP allosteric effectors by
anion-exchange high performance liquid chromatography (HPLC).
The concentration of the vaccinia and mouse R2 proteins used in these
experiments was 4 µM. Vaccinia R1 was added to 1 µM final concentration. ATP and DTT were present in all
reaction mixtures at 2.5 and 50 mM, respectively. The rNDP
substrates were added either at equimolar (0.15 mM each) or
in vivo concentrations. The in vivo
concentrations for rCDP, rUDP, rADP, and rGDP were set to 50, 250, 600, and 100 µM, respectively. These values were based on the
experimentally determined molar ratio of rNDPs measured in
vaccinia-infected BSC40 cells (see below). The in
vivo dNTP concentrations used in these assays were 15, 10, and 5 µM dATP, dTTP, and dGTP, respectively. These
concentrations were based on dNTP measurements made in
vaccinia-infected BSC40 cells (12). The volume of a
vaccinia virus-infected BSC40 cell was not determined in
these experiments. However, nucleated mammalian cell volumes range from
approximately 1 to 3 picoliters per cell (13). At the mid-range value
of 2 picoliters, the estimated concentrations of dATP, dTTP, and dGTP
are the values given above and used in these experiments. Although
microcompartmentation effects might affect these values, the fact that
vaccinia virus replicates in the cytoplasm of infected cells indicates
that the dNTPs are distributed throughout most of the aqueous portion
of the intracellular space.
Measurement of rNDP Levels in Vaccinia Virus-infected
BSC40 Cells--
Monkey kidney BSC40 cells
were infected with vaccinia virus strain WR as described previously (8,
12). Cells were harvested 4 h postinfection so that the nucleotide
pool measurements would reflect the levels that exist during peak viral
DNA replication. Cell extracts were prepared by treating cells with 0.4 M aqueous perchloric acid. After 5 min on ice, the
perchloric acid-extracted cells were pelleted by centrifugation, and
the supernatant was neutralized with NaOH (neutralization with KOH
resulted in a significant loss of nucleotides from the soluble
fraction). The extract was lyophilized to dryness, and the residue was
redissolved in 75 mM ammonium phosphate buffer (pH 3.7)
just prior to analysis. The total nucleotide extract was analyzed by
HPLC, using the same anion exchange column and ammonium phosphate
buffer system used in the four-substrate RNR assay. Peak identities
were verified by "spiking" the extract with authentic standards.
Ribonucleotide Reductase Activities in the Presence of the General
Allosteric Effector ATP--
Fig. 1
demonstrates the application of the four-substrate RNR assay to the
purified vaccinia virus enzyme. In this experiment, ATP (2.5 mM) was added as the sole allosteric modifier. Under these
conditions, CDP and GDP were the principal substrates reduced. Reduction of UDP was low or negligible in this and all other assays performed. Mammalian cells have been shown to derive most of their DNA
thymidylate residues from deoxycytidine nucleotides, via the dCMP
deaminase reaction, rather than through the reduction of uridine
nucleotides (14), and these data suggest that the same situation holds
true for vaccinia virus-infected cells. In this experiment, the total
RNR activity, summed for the four substrates, was 77 nmol/min/mg of R1
protein. This value corresponds to a turnover number of 12.4 min-1, comparable to values reported for the recombinant
mouse RNR (10, 15).
Regulation of the Vaccinia RNR by Nucleoside Triphosphate
Effectors--
Most of our present understanding of the allosteric
regulation of mammalian rNDP reductase is based upon analysis of the
effects of one or two modifiers rNDP Concentrations in Vaccinia Virus-infected BSC40
Cells--
Our application of the four-substrate assay to T4
phage-coded aerobic ribonucleotide reductase indicated that
intracellular ribonucleoside diphosphate concentrations constitute a
significant regulatory factor. The relative rates of the four reactions
corresponded closely to the nucleotide composition of T4 DNA, but only
under conditions where all four nucleoside triphosphate effectors and all four nucleoside diphosphate substrates were present at their approximate intracellular concentrations, as estimated from pool size
measurements. In determining whether the vaccinia rNDP reductase behaves similarly, it was first necessary to measure ribonucleoside diphosphate pool sizes in vaccinia virus-infected cells. These values,
determined by HPLC measurements of virus-infected cell extracts as
described under "Materials and Methods," are presented in Table
II. The approximate concentrations
reported fall within the ranges of values compiled by Traut (16) for
uninfected mammalian cell cultures. It should be observed that the HPLC
peaks from which these pool sizes were determined almost certainly
include deoxyribonucleoside diphosphates, as well as the
ribonucleotides. However, the data compiled by Traut (16) indicate that
in mammalian cells, the rNDP pools exceed those of dNDPs by
100-1000-fold. Therefore, we assumed the contribution to peak area by
the dNDPs to be negligible.
To estimate the in vivo molar concentration of each rNDP
from the pool size values in pmol/106 cells, we used 2 picoliters per cell, a mid-range value for the compiled volumes of
cultured mammalian cells, as reported by Hauschka (13). These estimated
concentrations, listed in Table II, are quite close to average
concentrations of rNDPs in a range of mammalian cells (16).
Effects of Natural Nucleotide Pool Asymmetries upon dNDP Product
Profiles--
As noted earlier, the T4 aerobic rNDP reductase in
vitro reduces the four substrates at rates corresponding to the
nucleotide composition of T4 DNA, but only under conditions where both
substrates and allosteric effectors are present at their approximate
intracellular concentrations. In mammalian cells, the ribonucleoside
diphosphate pools are more strikingly asymmetric than in T4
phage-infected Escherichia coli, with more than an order of
magnitude variation between the most (ADP) and least (CDP) abundant
(Table II and Ref. 16), whereas the corresponding asymmetry in the
bacterial system is less than 3-fold (17). Table
III summarizes results of assays carried
out under different conditions of substrate and effector concentrations
(equimolar substrates, in vivo substrates, no dNTP
effectors, in vivo effectors, etc.). Note that only in the
presence of in vivo levels of both effectors and substrates does the product profile approach the nucleotide composition of the
vaccinia virus genome. In particular, ADP reduction, which is
negligible in the absence of dNTP effectors, approaches the representation of dATP in the genome under "physiological"
conditions. Also, CDP reduction, which dominates the profile in the
presence of equimolar substrates and no dNTPs, is brought to a
reasonable level (34% of total activity) under physiological
conditions. Note that 34% is still twice the representation of dCMP in
the vaccinia genome. However, as noted earlier, dCMP is a major
contributor to the dTTP pool, via the dCMP deaminase reaction. The sum
of the CDP and UDP reductase activities under physiological conditions, 46%, is close to the representation of dCMP plus dTMP in the vaccinia genome (50%).
Effects of Proportional Variations in rNDP Substrate
Concentrations--
The data of Table III show one significant
discrepancy between rNDP substrate specificity in vitro,
under assumed biological conditions, and presumed reaction flux rate
in vivo. dGMP accounts for only 17% of vaccinia DNA
nucleotides, whereas the GDP reductase flux under physiological
conditions represented 26% of the total. This result was somewhat
surprising, because dGTP is by far the least abundant of the four dNTPs
in cell-free extracts, both of uninfected and vaccinia virus-infected
mammalian cells (12, 18); hence, we might have expected low flux rates
through rNDP reductase to contribute toward the underrepresentation of
dGTP in the dNTP pool. However, because of uncertainties in the cell volumes upon which we based our rNDP concentration estimates, as well
as intracellular factors that might alter effective nucleotide concentrations, the rNDP concentrations listed in Table II could be in
error by 2-fold or more.
On the other hand, it seems likely that any error affecting the
concentration estimate for one nucleotide would affect the other three
in proportion. For example, if the concentration of GDP were
overestimated by 2-fold, it seems likely that concentrations of the
other three rNDPs would be overestimated by similar factors. Thus, we
believe that the molar ratios calculated from rNDP pool data (second
column in Table II) accurately reflect the relative concentrations of the four substrates sensed by rNDP reductase in
vivo.
Accordingly, we prepared a "bioproportional" substrate mixture, in
which the four rNDPs were present at their molar ratios as determined
from pool analyses (ADP, CDP, GDP, and UDP present at a ratio of
12:1:2:5, respectively). This mixture was used for substrate saturation
experiments, with concentrations of the individual substrates varied,
but always in proportion to the representation of each rNDP in the
bioproportional mixture. Results of two separate experiments are
plotted together in Fig. 4. We found UDP
and CDP to behave according to Michaelis-Menten kinetics, with
saturation reached or approached at the highest concentrations tested.
However, ADP and GDP both showed inhibition at the highest
concentrations tested. The effect of increased concentration upon ADP
reduction was particularly striking, with the highest rate of ADP
reduction seen at the lowest concentration.
The small but significant decrease in GDP reduction at the highest rNDP
concentrations could mean that true intracellular concentrations sensed
by RNR are somewhat higher than those estimated in the experiment of
Table II. Thus, in vivo flux rates for vaccinia virus rNDP
reductase may conform even more closely to the nucleotide composition
of vaccinia DNA than does the enzyme in the in vitro experiment of Table III. However, of greater interest is the mechanism by which purine rNDP reduction, particularly that of ADP, evidently becomes inhibited at higher concentrations. Note that because of its
high representation among the rNDPs, ADP reduction rates are quite high
at the lowest levels tested, because it is probably close to saturating
the enzyme under these conditions. This factor
But is ADP the actual inhibitory species? In principle, inhibition
could be brought about by the product, dADP, because
deoxyribonucleoside diphosphates have been reported to act as rNDP
reductase regulators under some conditions (19). This seems unlikely,
because reaction rates were constant over the interval of each
experiment (data not shown). Had dADP accumulation been responsible for
the inhibition, then its progressive accumulation would be expected to
progressively slow down the rates of ADP and GDP reduction. Moreover,
the smallest amounts of dADP were formed in the reaction mixtures
showing the greatest inhibition.
Alternatively, if nucleoside diphosphate kinase were present in the
reaction mixtures, the dADP formed could be converted to dATP. This
possibility can be ruled out, first, because dATP accumulation would
inhibit all four activities, not just those on purine substrates, and
second, because our vaccinia R1 and R2 preparations have no detectable
NDP kinase activity (which would have been detectable in our assay
system). This can be seen from the data of Fig. 2, where the peak area
for each dNTP tested as an allosteric modifier corresponds closely to
the concentration added to the reaction mixture at time zero. Had NDP
kinase been present, the amounts of dNTPs would have increased over the
time of incubation. The ability to detect a number of potential
contaminating enzyme activities represents an additional strength of
the four-substrate assay procedure.
Although limited, the data suggest that inhibition of ADP and GDP
reduction at high rNDP concentrations is brought about by ADP itself.
This was tested in the experiment shown in Fig.
5, in which the concentration of ADP
alone was varied in the presence of constant amounts of CDP (50 µM) and GDP (100 µM). CDP reduction was
unaffected over the ADP concentration range tested (50-600 µM), whereas the reduction of both purine nucleotide
substrates was substantially inhibited. We have not yet asked directly
whether GDP is inhibitory, but if both ADP and GDP are inhibitors, the data shown in Fig. 4 suggest that the effect of ADP is considerably more pronounced.
Activity of a Chimeric RNR Holoenzyme--
From the above, it is
clear that an important goal is to learn whether rNDP reductase
inhibition by ADP is a general regulatory feature of this enzyme, by
carrying out kinetic experiments of the kind described here with a
mammalian form of the enzyme. As noted earlier, the vaccinia virus
enzyme represents a good model for the mammalian RNR. We had an
opportunity to test this concept further, because of the availability
of mouse R2 protein, purified as a recombinant protein from an
overexpressing clone furnished by Dr. Lars Thelander (Umeå
University). It was a simple matter to ask whether this protein could
function effectively with the vaccinia virus R1 protein in a
four-substrate assay. As shown in Fig. 6,
the answer was yes. Under the reaction conditions tested (2.5 mM ATP, no dNTP effectors, equimolar rNDP mix at 0.15 mM each), the reduction of UDP and GDP was negligible, but
the activities on ADP and CDP were considerable, with the specific
activity from the reduction of all four substrates amounting to 48 nmol
dNDP·min
In evaluating this experiment, we must consider the possibility that
the activity results from E. coli R1 and R2 proteins, which
are present as minor contaminants in our recombinant vaccinia and mouse
protein preparations. Three observations rule against this possibility.
First, in the experiment shown in Fig. 6, neither subunit had
detectable activity when tested alone (data not shown). Second, the
dATP-Sepharose column protocol used to purify vaccinia R1 protein is
specifically designed to remove any E. coli R1. Vaccinia R1
requires 75 mM ATP for elution from the column, whereas E. coli R1 is efficiently eluted at 5 mM ATP;
therefore, our procedure involved a 5 mM ATP elution step
before desorption of the vaccinia R1 at 75 mM ATP elution.
Third, in experiments not shown, we have run a four-substrate assay on
recombinant E. coli R1-R2 complex under conditions identical
to those of Fig. 6. The product profile was quite different, with the
order of activities for the E. coli enzyme being A > G > C > U. By contrast, the chimeric enzyme depicted in
Fig. 6 showed relative activities C > A > U > G.
Allosteric Regulation of Vaccinia Virus Ribonucleotide
Reductase--
The principal aim of this study was to apply the
four-substrate ribonucleotide reductase assay to an understanding of
the metabolic regulation of a eukaryotic form of the enzyme, using the
vaccinia virus RNR as a convenient model for the mammalian cell enzyme.
The viral enzyme behaved identically to mammalian RNRs, as shown
previously in single-substrate assays (1, 2, 20), in its response to
individual dNTPs. As noted in Table I, dCTP had no discernible effect
on any of the four reactions, whereas dATP inhibited all four to about
the same extent and at concentrations shown to have similar effects on
the mammalian enzyme. For example, the calf thymus enzyme was earlier
found to have 43% of the control activity for CDP reduction at 10 µM dATP (20), a value very close to that suggested from
Fig. 3 for the four-substrate assay of the vaccinia enzyme. The
specific stimulatory and inhibitory effects of dGTP and dTTP are also
identical to those noted previously for the mammalian RNRs. Presumably, all of these effects involve binding to activity and specificity sites,
as has previously been described for the mammalian enzyme (1, 2). The
total activity (sum of the four individual activities) was highest in
the presence of ATP alone, the general allosteric activator. The
turnover number determined, 12.4 min-1, was somewhat lower
than values determined from single-substrate assays for either the
recombinant vaccinia enzyme (18 min-1) (9) or the
recombinant mouse enzyme (24 min-1) (15). However, data
from our laboratory (21) indicate that the vaccinia RNR is present in
significant excess over its need to provide precursors for viral genome
replication at observed rates, even when assayed with the
nonphysiological electron donor dithiothreitol.
However, the individual effects upon enzyme specificity of a partial
recreation of intracellular conditions will be much harder to discern.
The data of Table III clearly establish the importance of running
reactions in the presence of physiological concentrations of both
nucleoside diphosphate substrates and nucleoside triphosphate effectors. The individual regulatory effects involved include an
undoubtedly complex mixture of the following: allosteric interactions of ATP and dNTPs at activity and specificity sites, competition among
the four rNDP substrates for binding to catalytic sites, product
inhibition by dNDPs, possible interactions of both ribo- and
deoxyribonucleoside diphosphates with activity and specificity sites
(19, 22, 23), and (possibly related to those interactions) the apparent
inhibition of purine rNDP reduction by ADP. Previous studies have
indicated that mammalian ribonucleotide reductase activities are
influenced by both rNDP substrates and dNDP products (19, 22, 23). In
some, but not all, of these earlier studies, inhibition by rNDPs was
competitive, as expected if alternative substrates are competing for
binding to catalytic sites. However, in some studies, the inhibitory
effects of alternative rNDP substrates were found to be noncompetitive
(22, 23), suggesting that binding occurs at a location other than the
catalytic site. One of these studies (22) was carried out with the
herpes simplex type 1 rNDP reductase, an enzyme that is distinctive in
being largely free of allosteric control modulated by nucleoside
triphosphates. The data suggested that regulation with this enzyme is
accomplished by a mixture of competitive binding of substrates and
inhibition by dNDP products, both effects occurring at catalytic sites.
From existing data (19, 22, 23), it is evident that deoxyribonucleoside
diphosphates function not only by product inhibition, but similarly to
their triphosphate counterparts, presumably by interaction with
allosteric sites. Data on dNDP pool sizes, necessary to establish the
biological significance of these effects, are sparse. However, those
data that do exist (16) suggest that under most circumstances, dNDP
levels are far lower than those shown to have significant allosteric
effects in vitro. The pool sizes of ribonucleoside
diphosphates are much higher, but the complexity of considering
catalysis, binding to catalytic sites, competition with alternative
substrates for binding, and possible interaction with allosteric or
still undefined sites indicates that physical studies of nucleotide
binding will be needed to understand the regulatory effects of rNDPs.
dNDP Product Profile vis-à-vis the Genomic Nucleotide
Composition--
Because ribonucleotide reductase participates in
synthesis of all four DNA precursors and because deoxyribonucleoside
triphosphates have no known roles except as DNA precursors, it is easy
to see why the enzyme should have regulatory mechanisms to ensure that the four deoxyribonucleotides are synthesized at rates proportional to
their representation in the genome. The four-substrate assay allows us
to see that the enzyme actually does function that way, when provided
with substrates and effectors at their approximate intracellular
concentrations. For the vaccinia enzyme, the most significant deviation
from this pattern involved the reduction of GDP. Although dGMP
represents just 17 mol % of vaccinia virus DNA, the GDP reduction rate
was 26% of the total rate (Table III) under simulated intracellular
conditions. Under similar conditions, the T4 phage rNDP reductase,
which also serves an AT-rich genome, reduced GDP at just 12% of the
total rate (4), suggesting an interesting distinction between these two
forms of RNR. However, the two reductases are similar in that
presenting the enzyme with an asymmetric substrate mixture, which
represents in vivo pool asymmetries, is essential if the
enzyme is to behave as it evidently behaves within the cell, producing
DNA precursors at rates corresponding to the nucleotide composition of
the genome. It will be of interest to ask whether ribonucleotide
reductases that serve GC-rich genomes also produce deoxyribonucleotides
in proportion to the base composition of the genome.
Our observations led us to carry out substrate saturation experiments
with a bioproportional rNDP mixture, varying the concentrations of all
four substrates in proportions that represented the relative pool sizes
of these four nucleotides. This led in turn to our finding that GDP
reduction rates decrease at higher substrate concentrations, suggesting
that relatively small adjustments in substrate concentrations would
allow us to approach even more closely a product profile that truly
represents vaccinia virus DNA base composition.
Substrate Inhibition of Ribonucleotide Reductase
Activity--
However, the most interesting result of our
bioproportional substrate saturation experiments was the significant
inhibition of ADP reduction by ADP itself and, at higher
concentrations, the inhibition of GDP reduction. To what site or sites
is ADP binding in order to achieve this effect? Although the
conventional wisdom of RNR allostery presumes that only the
triphosphates bind the allosteric sites, it seems reasonable that
molecules as similar in structure as ADP and ATP, for example, could
bind the same nucleotide binding site. In the bioproportional mixture,
ADP is the most abundant substrate by as much as 12-fold. This biased rNDP ratio may facilitate the preferential binding of ADP to an allosteric site. In most forms of RNR, binding of the
adenine-containing nucleotides to the specificity site stimulates
reduction of the pyrimidine rNDPs at the expense of the purine
substrates. If binding of ADP to the specificity site results in
similar allosteric behavior, then the expected outcome would be
stimulation of pyrimidine reduction at the expense of ADP reduction.
Therefore, as the concentration of ADP is increased, the rate of ADP
reduction should decrease relative to the reduction of the other
substrates. This hypothesis is also consistent with the inhibition of
GDP reduction observed at higher levels of the bioproportional mixture.
Testing the hypothesis, however, will require physical studies of ADP
binding to the R1 protein.
Is the inhibition by ADP physiologically significant? Because the
effect is seen in vitro at nucleotide concentrations
corresponding to in vivo levels, that seems a reasonable
supposition. Possibly this could represent a mechanism to prevent the
accumulation under certain circumstances of dATP, which has been shown
at low concentrations in vitro to stimulate activations of
proteases in a cascade of events leading to apoptosis (24) and which is
also a potent inhibitor of DNA replication through its effect on
ribonucleotide reductase. Inhibition by ADP could also help explain the
phenomenon explored in the companion paper, namely, the specific
inhibition of ADP reduction in vivo by hydroxyurea (11). If
hydroxyurea inhibition of RNR causes all four rNDPs to accumulate in
the short term, then the accumulation of ADP, the most abundant rNDP,
could lead to more specific inhibition of ADP reductase in the longer term, with a consequent specific depletion of dATP pools.
The Vaccinia-Mouse RNR Chimera--
Although not related to the
central theme of this investigation, it was interesting to learn that a
hybrid form of rNDP reductase, containing vaccinia R1 and mouse R2, had
enzymatic activity comparable to that of wild-type forms of the enzyme.
Previous attempts to generate active chimeras of the T4 and E. coli RNRs had revealed no activity (25).
When one considers what is known about the interactions between R1 and
R2, the activity of a mouse-vaccinia chimera may not be so surprising.
The large and small subunits of the class I RNRs interact primarily
through highly specific contacts formed between the R2 carboxyl
terminus and its corresponding binding site located on the R1 protein.
The R2 C-terminal sequences are not conserved but are highly
species-specific. But these sequences in the mouse and vaccinia
proteins are closely related, as shown by the following comparison of
the 10 C-terminal residues of mouse and vaccinia R2 proteins.
*
This work was supported by National Science Foundation Grant
MCB 96 06384 and National Institutes of Health Grant GM-55234.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Dept. of Biochemistry
and Biophysics, Oregon State University, 2011 Agricultural and Life
Sciences Bldg., Corvallis, OR 97331-7305. Tel.: 541-737-1865; Fax:
541-737-0481; E-mail: mathewsc{at}ucs.orst.edu.
The abbreviations used are:
RNR, ribonucleotide
reductase; dNDP and dNTP, deoxyribonucleoside 5'-di- and -triphosphate; rNDP, ribonucleoside 5'-diphosphate; HPLC, high performance liquid chromatography.
Allosteric Regulation of Vaccinia Virus Ribonucleotide Reductase,
Analyzed by Simultaneous Monitoring of Its Four Activities*
and
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
ATP, dATP, dGTP, and dTTPsimilar to those previously
reported for rNDP reductase of mouse, which the viral enzyme closely
resembles. This investigation uses the vaccinia enzyme as a readily
available and convenient model for understanding the cellular enzyme.
As previously reported for T4 phage aerobic rNDP reductase, we found the relative activities of ADP, CDP, GDP, and UDP reduction to be
reasonably close to the proportions of the four deoxyribonucleotides in
the vaccinia virus genome, but only when the four substrates and the
four allosteric effectors were all provided at their approximate intracellular concentrations. GDP reductase levels were somewhat higher, proportionately, than the representation of dGMP in vaccinia virus DNA. To understand this behavior and also to evaluate possible relationships between ribonucleotide reductase control and the very low
dGTP pools seen in eukaryotic cells, we carried out substrate saturation experiments with a "bioproportional" mixture containing the four rNDP substrates at their relative in vivo
concentrations as determined from rNDP pool measurements. Reduction of
the two purine substrates was inhibited at high concentrations of this mixture, and data suggest that ADP acts as a specific inhibitor of its
own reduction and that of GDP. Use of the four-substrate assay revealed
also that a mixture of vaccinia virus R1 protein and mouse R2 protein
is catalytically active, making this the first reported chimeric rNDP
reductase to show biological activity.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
2
2
tetrameric structure characteristic of the cellular enzymes. Also, like
the cellular RNRs, the viral enzyme reduces ribonucleoside diphosphates to the corresponding deoxyribonucleotides, via a free radical mechanism
that involves a tyrosyl residue on the R2 subunit (5), with catalytic
and allosteric sites located on the R1 subunit. The vaccinia R1 and R2
proteins share about 80% amino acid sequence identity with
corresponding mammalian RNR proteins (6, 7). Preliminary data, obtained
when the vaccinia enzyme was first described in our laboratory (8),
indicate that the viral enzyme is regulated similarly to the mammalian
enzyme. This study explored that regulation in more detail, using the
four-substrate assay.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-D-galactopyranoside-inducible pET
expression vectors and were overexpressed as described (5, 9). The
mouse R2 expression system (10) was a gift from the laboratory of Lars
Thelander (Umeå University, Umeå, Sweden). Overexpression of the
mouse R2 was carried out according to the procedure used to express the
vaccinia R2. The mouse R2 clone contains two selectable markers
encoding drug resistance gene products, and therefore, the addition of
25 µg/ml chloramphenicol was required in conjunction with 100 µg/ml ampicillin.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
View larger version (25K):
[in a new window]
Fig. 1.
Activities of vaccinia ribonucleotide
reductase in the presence of ATP as the sole allosteric effector,
measured with the four-substrate assay. The reaction mixture
contained 1 µM R1, 4 µM R2, 2.5 mM ATP, and 0.15 mM each of ADP, CDP, GDP, and
UDP. Aliquots of 100 µl each were removed from the reaction mixture
at 4-min intervals, subjected to boronate chromatography, and analyzed
by HPLC as described under "Materials and Methods."
typically, an activity site effector
(dATP or ATP), in the presence or absence of a specificity site ligand (ATP, dATP, dGTP, or dTTP)upon the reduction of single substrates. To
compare the vaccinia virus rNDP reductase with its mammalian counterpart, we determined the effect of each of the four dNTPs upon
the four activities of the virus enzyme in the presence of ATP, in
comparison with an assay carried out in the presence of ATP alone. As
shown in Fig. 2, the viral enzyme behaves
virtually identically to the reported behavior of mammalian rNDP
reductases. dCTP has no significant effect on any of the activities,
whereas dTTP activates GDP reduction and inhibits reduction of ADP and CDP. dGTP activates reduction of ADP and inhibits CDP reduction, whereas dATP, at the concentration used, inhibits all four reactions. In this experiment, the percentage of inhibition of the reduction of
each substrate by dATP (relative to the control in Fig. 2A) was 36% for ADP, 38% for CDP, 46% for GDP, and 40% for UDP. These effects are summarized in Table I. Other
experiments (not presented) showed CDP reduction to be stimulated by
dATP, when added in the absence of ATP, consistent with the action of
dATP as a positive effector for CDP reduction when bound at the
specificity site. At higher concentrations, the inhibitory effect of
dATP (in the presence of ATP) increased, as shown in Fig.
3. Within the likely physiological dATP
concentration range (5-15 µM), the activity (sum of the
four activities) was inhibited by 25-50%, values comparable to those
reported for single substrates with the mammalian enzymes. These data
confirm the appropriateness of using vaccinia rNDP reductase as a model
for understanding cellular eukaryotic forms.
View larger version (17K):
[in a new window]
Fig. 2.
Regulation of vaccinia virus ribonucleotide
reductase activities by individual dNTPs. All reaction mixtures
contained ATP at 2.5 mM and all four rNDP substrates at
0.15 mM each. Individual dNTP effectors were added to a
final concentration of 10 µM. Reaction mixtures were
incubated at 37 °C for 6 min.
Regulation of vaccinia virus ribonucleotide reductase activities by
single dNTPs
View larger version (10K):
[in a new window]
Fig. 3.
Inhibition of vaccinia virus RNR by
dATP. This figure shows the total RNR activity (sum of the four
activities) remaining after addition of dATP to the reaction mixture.
The decrease in activity was calculated by comparing the total amount
of rNDP reduced in the presence of 2.5 mM ATP with the
amount reduced when the reaction mixture contained 2.5 mM
ATP plus dATP at the indicated concentration.
Ribonucleoside diphosphate concentrations in vaccinia virus-infected
BSC40 cells
Effects of variations in substrate and effector concentrations upon
specificity of vaccinia ribonucleotide reductase in the four-substrate
assay
View larger version (15K):
[in a new window]
Fig. 4.
Reaction velocity versus
substrate concentration curves determined with a bioproportional
mixture of rNDPs. Each data point represents an
activity determined in a four-substrate assay run in the presence of
2.5 mM ATP and dNTPs at in vivo concentrations.
Incubation time for each assay was 5 min, an interval within which each
reaction rate remained constant. Results of two experiments are
plotted, with open circles denoting the results of the first
experiment and closed circles depicting the second
experiment.
the abundance of
ADPcould make it advantageous for the ADP reduction flux to be
limited at the higher concentrations needed for the enzyme to act
efficiently upon the other three rNDPs. In particular, this could be
seen as a factor limiting the accumulation of dATP, which could
otherwise block DNA replication through activity site binding to rNDP
reductase, with consequent depletion of the pools of dCTP, dGTP, and dTTP.
View larger version (20K):
[in a new window]
Fig. 5.
Effect of ADP concentration upon reduction of
ADP, CDP, and GDP in a three-substrate reaction mixture.
Conditions were identical to those of the experiment of Fig. 5, except
that only the concentration of ADP was varied. CDP and GDP
concentrations were held constant at 50 and 100 µM,
respectively.
1·mg R11, which corresponds to
a turnover number of 7.6 min1. This is similar to the
turnover number determined for the native vaccinia R1-R2 complex (12.4 min-1, determined in the experiment shown in Fig. 1).
However, the specificities of the two enzymes are quite different under
these reaction conditions; in particular, GDP is a good substrate for the native enzyme but is hardly touched by the chimeric enzyme.
View larger version (23K):
[in a new window]
Fig. 6.
Ribonucleotide reductase activity of a
vaccinia R1-mouse R2 chimera. In this experiment, a
reaction mixture containing 1 µM vaccinia R1, 4 µM mouse R2, 2.5 mM ATP, and equimolar
substrates (0.15 mM each) was sampled at 3-min
intervals.
DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
The underlined residues represent amino acids in the vaccinia R2
protein that differ from those of the mouse (which is identical to the
human sequence). All of the differences except one represent conservative substitutions. The activity of the chimeric enzyme suggests that the vaccinia enzyme is sufficiently closely related to
mammalian RNR to serve as a good model system. In addition, the
activity of a mouse R1-virus R2 chimera in vitro raises the interesting question of whether such enzymes are formed in
vivo, in vaccinia virus-infected cells. Although we might expect
the viral subunits to interact more strongly with each other than with
corresponding host cell proteins, such chimeras might form in infection
with viral mutants lacking either R1 or R2 proteins. Elsewhere, we have
discussed the question of whether such a complex might form in
vivo, in infection by a vaccinia virus mutant with a site-specific
deletion of the R1 gene (26). With the availability of recombinant
mouse R1 (15), the stage is set for asking whether this chimeric enzyme
actually can form in vitro.
FOOTNOTES
Present address: Dept. of Chemistry, Massachusetts Institute of
Technology 18-408, Cambridge, MA 02139.
REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  M. D. Hamilton, A. A. Nuara, D. B. Gammon, R. M. Buller, and D. H. Evans Duplex strand joining reactions catalyzed by vaccinia virus DNA polymerase Nucleic Acids Res., January 12, 2007; 35(1): 143 - 151. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. C. Magee, K. Y. Hostetler, and D. H. Evans Mechanism of Inhibition of Vaccinia Virus DNA Polymerase by Cidofovir Diphosphate Antimicrob. Agents Chemother., August 1, 2005; 49(8): 3153 - 3162. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. D. Hamilton and D. H. Evans Enzymatic processing of replication and recombination intermediates by the vaccinia virus DNA polymerase Nucleic Acids Res., April 20, 2005; 33(7): 2259 - 2268. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. P. Hendricks and C. K. Mathews Differential Effects of Hydroxyurea upon Deoxyribonucleoside Triphosphate Pools, Analyzed with Vaccinia Virus Ribonucleotide Reductase J. Biol. Chem., November 6, 1998; 273(45): 29519 - 29523. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Andersson, M. Westman, A. Hofer, and B.-M. Sjoberg Allosteric Regulation of the Class III Anaerobic Ribonucleotide Reductase from Bacteriophage T4 J. Biol. Chem., June 23, 2000; 275(26): 19443 - 19448. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Chimploy and C. K. Mathews Mouse Ribonucleotide Reductase Control. INFLUENCE OF SUBSTRATE BINDING UPON INTERACTIONS WITH ALLOSTERIC EFFECTORS J. Biol. Chem., March 2, 2001; 276(10): 7093 - 7100. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Chimploy, M. L. Tassotto, and C. K. Mathews Ribonucleotide Reductase, a Possible Agent in Deoxyribonucleotide Pool Asymmetries Induced by Hypoxia J. Biol. Chem., December 8, 2000; 275(50): 39267 - 39271. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |