We have recently demonstrated that there are three such sites in murine R1 mR1 the specificity or s-site, the adenine or a-site, and the hexamerization or h-site [2,3], leading to a compl
Trang 1of mammalian ribonucleotide reductase
Jian He1, Be´atrice Roy2, Christian Pe´rigaud2, Ossama B Kashlan3and Barry S Cooperman1
1 Department of Chemistry, University of Pennsylvania, PA, USA
2 Laboratoire de Chimie Organique Biomole´culaire de Synthe`se, Universite´ Montpellier II, France
3 Department of Medicine, University of Pittsburgh, PA, USA
Ribonucleotide reductases (RRs, EC 1.17.4.1) form a
family of allosterically regulated enzymes that
cata-lyze the conversion of ribonucleotides to
2¢-deoxy-ribonucleotides and are essential for de novo DNA
biosynthesis and repair, regulating other enzymes in
the DNA synthesis pathway via control of the
nuc-leotide pool [1] Of the four known classes of RR
(Ia, Ib, II and III) class Ia, which requires two
dif-ferent subunits R1 and R2 for activity and catalyzes
the reduction of all four common NDPs, is the most
widespread, comprising all eukaryotic RRs as well as
some from eubacteria, bacteriophages and viruses
The R1 subunit contains the active site as well as
allosteric sites We have recently demonstrated that
there are three such sites in murine R1 (mR1) (the
specificity or s-site, the adenine or a-site, and the
hexamerization or h-site) [2,3], leading to a complex
pattern of regulation of enzymatic activity, the major features of which are summarized in Scheme 1, as follows: (a) ATP, dATP, dGTP, or dTTP binding to the s-site drives formation of R12; (b) ATP or dATP binding to the a-site drives formation of R14, which exists in two conformations, R14a and R14b, with the latter predominating at equilibrium; (c) ATP binding
to the rather low affinity (Kd 1–4 mm) h-site, which occurs at physiologically significant concentrations, drives formation of R16 – dATP does not bind to this site at physiologically significant concentrations; (d) the R22 complexes of R12, R14a, and R16 are enzymatically active, whereas the R22 complex of mR14b has little, if any, activity; and (e) the sub-strate specificity of RR is determined by the ligand occupying the s-site: ATP and dATP stimulate the reduction of CDP and UDP, dTTP stimulates the
Keywords
allosteric sites; enantioselectivity; L -ADP;
L -ATP; mammalian ribonucleotide reductase
Correspondence
B S Cooperman, Department of Chemistry,
University of Pennsylvania, Philadelphia,
PA 19104–6323, USA
Fax: 215 8982037
Tel: 215 8986330
E-mail: cooprman@pobox.upenn.edu
(Received 25 October 2004, revised 20
December 2004, accepted 7 January 2005)
doi:10.1111/j.1742-4658.2005.04557.x
Here we examine the enantioselectivity of the allosteric and substrate bind-ing sites of murine ribonucleotide reductase (mRR) l-ADP binds to the active site and l-ATP binds to both the s- and a-allosteric sites of mR1 with affinities that are only three- to 10-fold weaker than the values for the corresponding d-enantiomers These results demonstrate the potential of
l-nucleotides for interacting with and modulating the activity of mRR, a cancer chemotherapeutic and antiviral target On the other hand, we detect
no substrate activity for l-ADP and no inhibitory activity for N3-l-dUDP, demonstrating the greater stereochemical stringency at the active site with respect to catalytic activity
Abbreviations
mRR, mammalian ribonucleotide reductase; mR1, large subunit of mammalian ribonucleotide reductase; mR2, small subunit of mammalian ribonucleotide reductase; N3- D -dUDP, 2¢-azido-2¢-deoxy-b- D -uridine 5¢-diphosphate; N 3 - L -dUDP, 2¢-azido-2¢-deoxy-b- L -uridine 5¢-diphosphate;
RR, ribonucleotide reductase.
Trang 2reduction of GDP and dGTP stimulates the
reduc-tion of ADP dATP is a universal inhibitor of RR
activity due to its induction of R14b formation as a
result of a-site binding, whereas ATP is a universal
activator because it induces R16 formation as a
result of h-site binding
RR is a well-recognized target for cancer
chemo-therapeutic and antiviral agents [4–7], as illustrated by
the anticancer drugs hydroxyurea [8] and gemcitabine
[9,10] In recent years, l-nucleoside analogues have
been examined as novel therapeutic agents, which have
been shown to sometimes have comparable or higher
antiviral activity than their d-counterparts, as well as
more favorable toxicological profiles and superior
metabolic stability [11–15] Studies at the individual
enzyme level have shown that l-nucleosides and
l-nucleotides can bind to human or other mammalian
enzyme active sites (reviewed in [16,17]) and sometimes
act as substrates, in particular as phosphate donors
and acceptors, as is the case for l-ATP and
deoxycyti-dine kinase [18,19], l-nucleosides and deoxycytideoxycyti-dine
kinase, thymidine kinase 2 and deoxyguanosine kinase
[20], l-nucleoside-5¢-monophosphates and UMP-CMP
kinase [21], and, most recently
l-nucleoside-5¢-diphos-phates and phosphoglycerate kinase [22,23] In
contrast, almost no investigations of interactions of
l-nucleosides or l-nucleotides with allosteric sites have
been reported (although see [13]) In the present work,
we examine the abilities of l-ATP and of l-ADP
(Fig 1) to interact with the allosteric sites and active
site of mR1, respectively We also demonstrate that
mRR displays enantiospecificity with respect to the
b-d-configuration of the sugar moiety of
2¢-azido-2¢-deoxyuridine-5¢-diphosphate, paralleling our recent
results with Escherichia coli RR [24]
Results and Discussion
L-ATP is an allosteric effector of CDP reductase CDP is the only one of the four ribonucleoside diphos-phate substrates that is reduced by mRR with a signi-ficant activity in the absence of allosteric effectors, albeit with a high Km and a low kcat Addition of
3 mm d-ATP both lowers Kmand raises kcat[2] In the absence of d-ATP, CDP reductase activity shows a biphasic response to l-ATP, first increasing and then decreasing (Fig 2) Both the maximal observed activity and the concentration of l-ATP giving maximum activity vary with [CDP], with the first increasing and the second decreasing as [CDP] is increased In the presence of d-ATP, however, CDP reductase shows only a monotonic decline as a function of [l-ATP], with the apparent IC50 increasing only slightly as [d-ATP] is increased (Fig 3)
The effects of l-ATP on CDP reductase activity clo-sely mirror those obtained with dATP, albeit at much higher concentrations [2], and are well rationalized by Scheme 1 The results in Fig 2 provide evidence that, like dATP, l-ATP binds first to the s-site, inducing active R12 formation, and then to the a-site, inducing inactive R14b formation, thus accounting for the biphasic curves observed The changes in the curves in Fig 2 as a function of CDP concentration are consis-tent with the known induction of R12 formation by high levels of CDP, with the result that achieving max-imal activity requires lower [l-ATP] In contrast, we have previously shown that CDP reductase activity has
a triphasic response to increasing d-ATP concentra-tion, first increasing, then decreasing, then increasing again as d-ATP binds to the s-, a- and h-sites in sequence [2] High concentrations of l-ATP do not lead to a third phase activation of CDP reductase, leading to the conclusion that l-ATP, like dATP,
L-ATP
dATP
ATP
dGTP
dTTP
L-ATP dATP ATP
Scheme 1 Allosteric ligand effects on enzyme aggregation state
and activity [2,3] R2 has been omitted for simplicity – activity
assays were carried out in the presence of saturating [R2] States
shown with white text on dark background have high activity; those
on white background have little or no activity Inclusion of L -ATP is
based on results reported herein.
Fig 1 The structures of D - and L -adenine nucleotides.
Trang 3does not bind to the h-site, at least at concentrations
10 mm
With CDP as substrate, the dissociation constants of
l-ATP for the s- and a-sites, calculated as described
[2], are approximately 200 lm and 1000 lm,
respect-ively These values are three- to 10-fold higher than
the corresponding values for d-ATP (25 lm and
300 lm, respectively [2]), demonstrating the only
modest enantioselectivity at these two sites The
three-dimensional structure of mR1 has not yet been
determined However, its s-site and a-site are likely to
be very similar to those determined for the E coli class
Ia R1 (eR1 [25]), as almost all of the contact residues
in each of these sites in eR1 are conserved in mR1
(the s-site interactions are also largely conserved in known class Ib and class II structures [26–28]) It thus seems likely that the only modest loss in l-enantiomer affinity at these two sites reflects conservation of the known triphosphate and base interactions, with decreased affinity mainly resulting from loss of the interactions with the ribose, e.g in the s-site, between a specific Asp (D232 in eR1, D226 in mR1) and the ribose 3¢-OH
In the presence of 0.2 mm d-ATP (Fig 3a), mR1 should largely partition between active mR12, with
d-ATP bound to the s-site only, and inactive mR14b, with d-ATP bound to the s- and a-sites [2] Addition
of l-ATP should lead to saturation of the a-site and complete conversion to mR14b, leading to the observed monophasic inhibition By contrast, at higher d-ATP (3 mm, Fig 3c), d-ATP should be bound to all three sites, s-, a-, and h-, and mR1 should be mostly in the form of active mR16 [2] Earlier we showed that, under similar conditions, addition of dATP led to inhibition of activity, largely as a result of dATP displacement of d-ATP from the a-site, which weakens the binding of mR22 to mR16 [3] It is likely that the l-ATP inhibition seen in Fig 3c has a similar explanation
L-NDP interaction with the mR1 substrate site
An HPLC assay was used to determine whether
l-ADP could mimic d-ADP in being reduced by mRR
in the presence of the allosteric effector dGTP bound
to the s-site [2] As seen in Fig 4, approximately 11%
of d-ADP is converted to d-dADP when d-ADP is incubated for 2 h in the presence of mRR and dGTP
Fig 2 The effect of L -ATP on CDP reductase in the absence of
D -ATP Assays were carried out by preincubating 1.2 l M R1, 2.0 l M
R2, and varying concentrations of L -ATP for 7 min prior to
[8– 3 H]CDP addition.
Fig 3 The effect of L -ATP on CDP reductase in the presence of
D -ATP Activities were carried out as in Fig 2, but with a fixed
amount D -ATP in the preincubation buffer.
Fig 4 L -ADP is not a substrate for RR Samples were incubated for 2 h at 25 C in buffer A containing dGTP (300 l M ), R1 (0.84 l M ), R2 (4 l M ) and either (a) D -ADP (400 l M ) or (b) L -ADP (400 l M ) Quenched reaction mixtures were analyzed by HPLC Retention times: dGTP 25 ± 2 min ( L or D )-ADP 24 ± 1 min, D -dADP
30 ± 1 min The peaks at 29 ± 1 min and 33 ± 1 min are from
D -dAMP and oxidized dithiothreitol, respectively.
Trang 4By contrast, no conversion of l-ADP to l-dADP is
detectable under comparable conditions, or even
employing 14 times as much enzyme for periods up to
8 h (Fig 4B) These results lead to the conclusion that
the specific activity of mRR for reduction of l-ADP is
< 1% of that for d-ADP Similarly, under conditions
in which preincubation with the known suicide
inhib-itor N3-d-dUDP [29] at a concentration of 20 lm
redu-ces CDP reductase activity by 80%, incubation with
up to 250 lm N3-l-dUDP leads to no reduction in
activity (Fig 5) These results parallel those we
obtained with E coli RR [24]
Although the results in Figs 4 and 5 demonstrate the
failure of l-NDPs to act as a substrate or as a
mechan-ism-based inhibitor, l-ADP can clearly bind to the
substrate site, as shown by its inhibition of d-ADP
reduction (Fig 6) Measured in the presence of 2 mm
d-ATP, which is sufficient to saturate the a-site and
most of the h-site, l-ADP is a classic competitive
inhibitor of dGTP-dependent d-ADP reductase, with a
Ki of 1.0 mm, about 11-fold higher than the apparent
Km for d-ADP of 91 lm (Fig 6A,B) As above, this
probably reflects retention of the base and especially
b-phosphate interactions at the active site and loss of
the specific ribose interactions that are crucial for
sub-strate activation [25] l-ADP also displays competitive
inhibition in the absence of d-ATP (Fig 6C), but the
nonlinearity of the secondary plot (Fig 6D) is an
indica-tion of interacindica-tion at more than just the substrate site
One possibility is that, in the absence of d-ATP, l-ADP
not only acts as a competitive inhibitor, but also binds
to the otherwise empty a-site at high concentrations,
permitting it to act as an allosteric inhibitor as well Here we note that high concentrations of d-ADP induce mR14formation [2], providing evidence for NDP bind-ing to the a-site
L-Nucleosides as potential therapeutic agents directed against ribonucleotide reductase
We detect no substrate activity for l-ADP and no irre-versible inhibitory activity for N3-l-dUDP, demonstra-ting the considerable stringency at the active site of mR1 with respect to catalytic activity On the other hand, our results show that l-ADP binds to the active site and l-ATP binds to both the s- and a- allosteric sites of mR1 with affinities that are only three- to 10-fold weaker than the values for the corresponding
d-enantiomers, demonstrating the potential of l-nucleo-tides for interacting with and modulating the activity
of RR Particularly intriguing in this regard is the allosteric ligand dATP, which inhibits RR through a very high affinity interaction with the a-site (Kd, 0.3–
1 lm [2]) Efforts to explore the interaction with RR
of l-enantiomers of dATP and dATP analogues are currently underway
Experimental procedures
Materials
N3-d-dUDP and N3-l-dUDP were synthesized as described [24] l-AMP was obtained in 74% yield by selective phos-phorylation of l-adenosine with POCl3in triethylphosphate [30] After being converted to its tri-n-butylammonium salt,
l-AMP was first reacted with 1,1¢-carbonyldiimidazole
in 1,3-dimethyl-3,4,5,6-tetrahydropyrimidin-2(1H)-one and then with tri-n-butylammonium phosphate to give l-ADP
in 15% yield [31] l-ATP was obtained in 41% yield according to the Ludwig procedure [32] l-ADP and l-ATP were purified to homogeneity, as judged by HPLC and HRMS, by DEAE-Sephadex A-25 and RP18 chromatogra-phy, and converted to the respective sodium salts by passage through a Dowex-AG 50WX2-400 column The
l-nucleotides were fully characterized by NMR (1H, 13C,
31P), fast-atom-bombardment MS, UV spectroscopy and polarimetry l-ADP: [a]20
D+29 (c 1.0, H2O); 31P NMR (D2O, 300 MHz) d )8.2 (d, JP–P¼ 20.6), )10.8 (d, JP–P¼
(C10H14O10N5P2Na2), calculated 472.0011, found 471.9999
l-ATP: [a]20
D+22 (c 1.0, H2O);31P NMR (D2O, 300 MHz)
d )6.2 (d, JP–P¼ 18.2), )10.7 (d, JP–P¼ 18.2), )20.5 (t, JP–P ¼ 18.2); MS FAB+
m⁄ z 574 (M + H)+
; HRMS (C10H14O13N5P3Na3), calculated 573.9494, found 573.9453 Cloned murine R1 and R2 were prepared as described [33] Protein concentration was estimated by Bradford assay [34]
Fig 5 L -N3-UDP does not inactivate RR Solutions of 4 l M R1,
8 l M R2, and 2 m M D -ATP were preincubated in buffer A with the
indicated the amounts of N3- L -dUDP or N3- D -dUDP for 15 min (total
volume, 10 lL) Ninety microliters of [8- 3 H]CDP (278 l M ) and D -ATP
(2 m M ) in buffer B were then added, and reaction was allowed to
proceed for 30 min before quenching Reaction mixture analysis
was by phenyl boronate chromatography.
Trang 5using bovine serum albumin as standard [2,8-3H]ADP was
purchased from Perkin Elmer (Shelton, CT, USA), [8-3
H]-CDP, and [8-3H]-GDP was purchased from Amersham
(Pis-cataway, NJ, USA) Radioactive nucleotides were purified by
aminophenyl boronate agarose column chromatography
before use All the other nucleotides are from Amersham or
Roche (Mannheim, Germany)
Ribonucleotide reductase activity assay
RR was assayed by measuring the formation of [3H]dNDP
from [3H]NDP at 25C, as described [33] Reactions were
carried out in buffer A (50 mm Hepes, pH¼ 7.6, 10 mm
KCl, 10 mm MgCl2, 25 mm dithiothreitol, 7 mm NaF,
0.05 mm FeCl3) at 25C in a total volume of 100 lL, with
final protein concentrations (given as monomer) of mR1,
0.12–3.3 lm, and of mR2, 1.0–11 lm and the indicated
amount of nucleotides mRR was preincubated for 7 or
10 min prior to [3H]NDP addition Samples were quenched
(boiling water) after 10–40 min and analyzed by
phenyl-boronate-agarose chromatography Under these conditions, the amount of product formation was proportional to reac-tion time
HPLC analysis of RR-catalyzed reduction
Following quenching, reaction mixtures were centrifuged through a Millipore Microcon YM-10 centrifugal filter to remove proteins and filtrates were loaded onto a Higgins
C18 RP-HPLC analytical column The gradient was 0–2% (v⁄ v) acetonitrile in 20 min followed by 2–5% (v ⁄ v) aceto-nitrile in 20 min, containing 10 mm triethylammonium bicarbonate pH 7.7
Acknowledgements
This work was supported by NIH grant CA 58567 (BSC) and by the CNRS (BR) We thank Gilles Gos-selin for technical advice regarding l-nucleotide syn-thesis
Fig 6 L -ADP inhibition of dGTP-dependent ADP reductase Assay mixtures contained 0.21 l M R1, 1 l M R2, 100 l M dGTP, ± 2 m M D -ATP, variable amounts of [8- 3 H] D -ADP and the indicated amounts of L -ADP Preincubation time was 10 min prior to substrate addition Reaction was allowed to proceed for 40 min before quenching and analysis by phenyl boronate chromatography (A) Lineweaver–Burke plot of L -ADP inhibition of dGTP-dependent D -ADP reductase in the presence of 2 m M D -ATP (B) Secondary plot of the slope values of A (C) Lineweaver-Burk plot of L -ADP inhibition of dGTP-dependent D -ADP reductase in the absence of D -ATP (D) Secondary plot of the slope values of (C).
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