Class II Recombinant Phosphoribosyl Diphosphate Synthase from
Spinach
PHOSPHATE INDEPENDENCE AND DIPHOSPHORYL DONOR SPECIFICITY*
Britta N.
Krath and
Bjarne
Hove-Jensen
From the Department of Biological Chemistry, Institute of Molecular
Biology, University of Copenhagen, 83H Sølvgade, DK-1307 Copenhagen K,
Denmark
Received for publication, November 8, 2000, and in revised form, February 5, 2001
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ABSTRACT |
A recombinant form of spinach (Spinacia
oleracea) phosphoribosyl diphosphate (PRPP) synthase
isozyme 3 resembling the presumed mature enzyme has been synthesized in
an Escherichia coli strain in which the endogenous PRPP
synthase gene was deleted, and has been purified to near homogeneity.
Contrary to other PRPP synthases the activity of spinach PRPP synthase
isozyme 3 is independent of Pi, and the enzyme is inhibited
by ribonucleoside diphosphates in a purely competitive manner, which
indicates a lack of allosteric inhibition by these compounds. In
addition spinach PRPP synthase isozyme 3 shows an unusual low
specificity toward diphosphoryl donors by accepting dATP, GTP, CTP, and
UTP in addition to ATP. The kinetic mechanism of the enzyme is an
ordered steady state Bi Bi mechanism with KATP
and KRib-5-P values of 170 and 110 µM, respectively, and a Vmax
value of 13.1 µmol (min × mg of protein)
1.
The enzyme has an absolute requirement for magnesium ions, and maximal
activity is obtained at 40 °C at pH 7.6.
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INTRODUCTION |
The compound 5-phospho-D-ribosyl
-1-diphosphate (PRPP)1 is
a precursor in several important metabolic pathways, including purine, pyrimidine, and pyridine nucleotide de novo and
salvage synthesis, and histidine and tryptophan synthesis (1, 2). Certain microorganisms also utilize PRPP for the synthesis of methanopterin or polyprenylphosphate pentoses (3, 4). The synthesis of
PRPP is catalyzed by PRPP synthase: Rib-5-P + ATP
PRPP + AMP (5).
PRPP synthase is encoded by the PRS gene(s). Eukaryotic
organisms usually contain more than one PRS gene. Analysis of a cDNA library as well as genome sequencing have revealed the presence of at least five PRS genes in the flowering plant
Arabidopsis thaliana (Ref. 6,
GenBankTM/EBI database accession no. AC004521).
Spinach (Spinacia oleracea) appears to contain at least four
PRS genes, coding for PRPP synthase isozyme 1 to 4, all of
which have enzymatic activity. This was established by analysis of a
cDNA library for complementation of an Escherichia coli
prs allele (7). Spinach PRPP synthase isozyme 2 and
3 contain 76 and 87 additional amino acids at their N-terminal ends,
respectively, compared with spinach PRPP synthase isozyme 4 and PRPP
synthases from E. coli (8), Bacillus subtilis (9), or man (10). These transit peptides of the spinach PRPP synthases
contain consensus motifs for maturation of the polypeptides during
entry into organelles. Experimental evidence and amino acid sequence
comparison revealed that spinach PRPP synthase isozyme 2 was found
within the chloroplast, whereas isozyme 3 appeared to be located
within the mitochondrion and isozyme 4 in the cytosol. The location of
spinach PRPP synthase isozyme 1 is unknown. The deduced N-terminal
sequence of the presumed mature isozyme 3 polypeptide is
Asn-Ser-Val-Glu-Phe; the polypeptide contains 320 amino acids, and has
a calculated molecular mass of 35,497 Da. This value is similar to that
for PRPP synthases from most other organisms (7).
In the present work we report the properties of spinach PRPP synthase
isozyme 3. The enzyme was synthesized in E. coli and was
specified by a gene, which had been manipulated to make the gene
product resemble the presumed mature polypeptide, i.e.
without a transit peptide. Spinach PRPP synthase isozyme 3 and the
"classical" PRPP synthases from E. coli, B. subtilis,
and man differ greatly in their specificity for diphosphoryl donor,
dependence of Pi for activity, and in their allosteric
properties. We show that spinach PRPP synthase isozyme 3 belongs to a
novel class of PRPP synthases, Class II.
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EXPERIMENTAL PROCEDURES |
Materials--
Oligonucleotides were provided by Hobolth DNA
Syntese (Hillerød, Denmark). Restriction endonucleases were purchased
from Amersham Pharmacia Biotech, PRPP and Rib-5-P from Sigma, and
nucleotides from Roche Molecular Biochemicals.
[
-32P]dATP, [
-32P]GTP,
[
-32P]CTP, and [
-32P]UTP were
obtained from PerkinElmer Biosystems. [
-32P]ATP was
prepared as previously described (11). Other chemicals were from Sigma
or Merck.
Bacterial Strains and Growth Media--
The E. coli
strains used were XL1-Blue (araD
(ara-leu) galE
galK
(lac)
74 rpsL hsdR
(rK
mK
) mcrA mcrB/F
lacIq zzf::Tn10)
(Stratagene), HO773 (araCam araD
(lac)U169 trpam malam rpsL relA thi supF deoD gsk-3 udp
prs-4) (12) and HO1088, which is identical to HO773
except for the presence in the former strain of an F episome harboring
an lacIq allele as well as a Tn10
transposon. Episome transfer was performed by mixing exponentially
growing cultures of HO773 and XL1-Blue in a 50:1 ratio at 37 °C.
After incubation at 37 °C for 1 h leucine prototrophic-tetracycline resistant exconjugants were selected. Cells
were grown at 37 °C in NZY broth (13). When necessary the culture
medium was supplemented with ampicillin (100 mg liter
1),
tetracycline (10 mg liter
1), isopropyl
1-thio-
-D-galactopyranoside (5 µM), or NAD
(40 mg liter
1). Cell growth was monitored in an Eppendorf
PCP6121 photometer as absorbance at 436 nm. An optical density at 436 nm of 1 (1-cm path length) corresponds to ~3 × 1011
cells liter
1.
Manipulation of Spinach PRPP Synthase Isozyme 3-Specifying
cDNA--
To construct pBK862, DNA of pBK843 (7) was digested by
restriction endonucleases BstBI and EcoRV. The
resulting DNA fragments were subjected to electrophoresis in a 1%
agarose gel and the 1048-base pair DNA fragment containing
PRS3 was cut out and purified (Qiagen). Ten picomoles of
each of the oligodeoxyribonucleotides 5'-AATTCATTAAAGAGGAGAAATTAACTATGAATTCCGTCGAGTTTT (MNS) and
5'-CGAAAACTCGACGGAATTCATAGTTAATTTCTCCTCTTTAATG (MNS-komp) were annealed
by heating to 95 °C and slowly cooled to 0 °C. This annealing
resulted in the formation of a double-stranded DNA fragment with
EcoRI and BstBI overhangs. The underlined
nucleotides of the MNS oligodeoxyribonucleotide indicate the codons
specifying the N-terminal end of the presumed mature spinach PRPP
synthase isozyme 3 polypeptide. DNA of
pUHE23-22 was digested by
restriction endonucleases PvuII and EcoRI. The three DNA species were mixed and ligated by T4 DNA ligase (Promega), transformed (14) to E. coli strain HO1088 followed by a
selection for prs+ (2). The nucleotide sequence
of the resulting plasmid (pBK862) was confirmed by sequencing using an
Abi Prism 310 DNA Sequencer as recommended by the supplier (PerkinElmer).
Purification of PRPP Synthase and Protein Methods--
Two
liters of NZY broth supplemented with ampicillin, tetracycline, and
isopropyl 1-thio-
-D-galactopyranoside were inoculated with 100 ml of overnight culture of strain HO1088/pBK862 and incubated with shaking for 20 h. The following procedures were carried out at 4 °C. Cells (6 g of wet weight) were harvested by centrifugation (Sorvall, GS3 rotor) at 6000 rpm for 12 min, washed in 0.9% NaCl, collected by centrifugation (Sorvall, SS34 rotor), resuspended in 30 ml
of 50 mM Tris/HCl, pH 7.6 and homogenized for 6 ×1 min in
a Soniprep ultrasonic disintegrator (Measuring and Scientific Equipment, model 150). Debris was removed by centrifugation at 10,000 rpm for 15 min. Streptomycin sulfate (10% w/v in 50 mM Tris/HCl, pH 7.6) was added to a final concentration of 1%, and the
precipitate was removed by centrifugation at 10,000 rpm for 15 min. The
supernatant fluid was 45% saturated with solid
(NH4)2SO4. The precipitate was
removed by centrifugation, and the resulting supernatant fluid was 65%
saturated with solid (NH4)2SO4.
After centrifugation, the precipitate was dissolved in 50 mM Tris/HCl, pH 7.6, and an equal volume of a solution of
10% (w/v) polyethylene glycol 6000 was added. The precipitate was
collected by centrifugation, dissolved in 50 mM Tris/HCl,
pH 7.6 and applied to a column (1.0 × 25 cm) of Dyematrex Gel
Blue B (Millipore). After washing the column with three volumes of 50 mM Tris/HCl, pH 7.6 protein was eluted at 1 ml
min
1 with a 50-ml linear 0.0-0.5 M KCl
gradient in 50 mM Tris/HCl, pH 7.6. Fractions containing
PRPP synthase activity were combined and applied to a DE52 anion
exchange column (Whatman; 1.0 × 30 cm). Protein was eluted at 1 ml min
1 with a 100-ml linear 0.0-0.5
M NaCl gradient in 50 mM Tris/HCl, pH 7.6. Fractions containing PRPP synthase activity were combined, concentrated
by (NH4)2SO4 precipitation,
dialyzed against 50 mM Tris/HCl, pH 7.6 containing 50%
glycerol, and stored at
20 °C. The purity was evaluated by
SDS-PAGE and Coomassie Brilliant Blue staining (15). Protein content
was determined by the bicinchoninic acid procedure with chemicals
provided by Pierce (16). Bovine serum albumin was used as the standard.
Amino acid sequencing by automated Edman degradation was performed by
the Department of Biochemistry and Nutrition, Technical University of Denmark.
Assay of PRPP Synthase Activity--
PRPP synthase activity was
assayed at 37 °C as follows: Extract or enzyme (10 µl),
appropriately diluted, was mixed with 40 µl of a reaction mixture
(both prewarmed at 37 °C) containing radiolabeled
(deoxy)ribonucleoside triphosphate (1 kBq per assay), Rib-5-P,
MgCl2 or MnCl2, 20 mM NaF (omitted
after chromatography in DE52), 50 mM Tris/HCl, pH 7.6. Unless otherwise indicated the concentrations of ATP, Rib-5-P, and
MgCl2 (or MnCl2) were 2.0, 3.0, and 5.0 mM, respectively. Samples (10 µl) were removed at intervals and mixed with 5 µl of 0.33 M HCOOH. This 15 µl was applied to polyethyleneimine-cellulose plates (Baker-flex,
J. T. Baker) and dried. When [
-32P]ATP was used
as diphosphoryl donor, PRPP and ATP were separated by thin layer
chromatography in 0.85 M KH2PO4,
which had been previously adjusted to pH 3.4 with 0.85 M
H3PO4, as solvent. When [
-32P]dATP, [
-32P]GTP,
[
-32P]CTP, or [
-32P]UTP were used as
diphosphoryl donors, chromatography was performed as follows:
CH3OH (2 cm), 1 M CH3COOH (2 cm),
and 0.9 M CH3COOH-0.3 M LiCl (16 cm) (17, 18). By this procedure
[32P](deoxy)ribonucleoside triphosphate was separated
from [32P](deoxy)ribonucleoside monophosphate.
Radioactivity was quantitated in an Instant Imager (Packard, model 2024).
Kinetic Analysis--
Results of initial velocity determinations
and of product inhibition studies, both of which were the average of at
least three determinations, were fitted to the following equations
using the program UltraFit, (Biosoft, version 3.01). Equation 1 is the
Michaelis-Menten equation for hyperbolic substrate saturation kinetics,
whereas Equation 2 is the rate equation for a sequential Bi Bi
mechanism. For competitive inhibition, noncompetitive inhibition, and
substrate inhibition the initial velocities were fitted to Equations 3, 4, and 5, respectively (19, 20).
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(Eq. 1)
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(Eq. 2)
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(Eq. 3)
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(Eq. 4)
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(Eq. 5)
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where v is the initial velocity,
Vapp is the apparent maximal velocity,
Km is the apparent Michaelis
Menten constant for
the varied substrate S, Vmax is the maximal
velocity, KATP and
KRib-5-P are the Michaelis
Menten constants for
ATP and Rib-5-P, respectively. KiATP is the
dissociation constant for ATP, Kis and
Kii are inhibitor constants for the inhibitor I
obtained from the effect on slopes and intercept, respectively, and
Ki is the inhibitor constant for the substrate S
(19, 20). Calculation of the free Mg2+ concentration was
performed as previously described (21).
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RESULTS |
Synthesis and Purification of Spinach PRPP Synthase Isozyme
3--
The DNA encoding the 87 codons specifying the presumed transit
peptide of spinach PRPP synthase isozyme 3 were removed and replaced by
an ATG triplet as described in "Experimental Procedures." Nucleotide sequencing of pBK862 as well as amino acid sequencing of the
purified recombinant PRPP synthase demonstrated its N-terminal amino
acid sequence to be Met-Asn-Ser-Val-Glu-Phe. This was identical to the
sequence of the presumed mature polypeptide with an additional methionine, which served as the translation initiation residue. Induction of expression of the resulting PRS3 allele,
harbored in pBK862, resulted in overproduction of the enzyme. PRPP
synthase was purified by (NH4)2SO4
and polyethylene glycol precipitations, followed by affinity
chromatography and anion exchange chromatography. The yield was 7.2 mg
of 95% pure enzyme with a specific activity of 13.1 µmol (min × mg of protein)
1.
Characteristics of Spinach PRPP Synthase Isozyme 3--
The enzyme
required the presence of Mg2+ for activity. When ATP and
Mg2+ were added at equimolar concentrations, the saturation
curve for ATP appeared sigmoid. When Mg2+ was added at 2 mM excess of ATP an increase in enzyme activity was
observed compared with the activity with equimolar concentrations of
ATP and Mg2+, and the saturation curve for ATP appeared
hyperbolic with a decrease in the Km for ATP. These
results indicate that Mg2+ was required as a free cation to
activate the enzyme and that the true substrate for the reaction was
MgATP. The enzyme accepted Mn2+ as an alternative cation
with an activity of 15% of that obtained with Mg2+. The
enzyme was inactive with the following divalent cations: Ca2+, Cd2+, and Co2+. The activity
of the enzyme was independent of Pi, and in addition no
activation by Pi was observed as the activity of the enzyme remained at 11 µmol (min × mg of protein)
1 at
Pi concentrations between 0 and 80 mM. The
effect of pH on the activity was determined in the range 6.5-9.0 with
Tris/HCl as buffer. The enzyme showed maximal activity at pH 7.6. The
activity increased steeply up to pH 7.2 and decreased with a shallower slope above pH 7.6. The shape of the curve for the dependence of
temperature on the activity was symmetrical with maximal activity at
40 °C. As a result of these analyses we chose to assay the activity
of the enzyme at 37 °C, at pH 7.6 and at a free Mg2+
concentration of at least 1.2 mM.
Kinetic Mechanism--
Initial velocity versus the
concentration of either ATP or Rib-5-P was found to follow typical
Michaelis-Menten kinetics. To determine whether the reaction occurred
by a sequential or a ping-pong mechanism, the effect of the ATP
concentration on the initial velocity was measured at different
concentrations of Rib-5-P and vice versa. In double reciprocal plots of
the data, intersecting lines indicated that the reaction followed a
sequential mechanism (Fig. 1). The data
were fitted to Equation 2 and resulted in a KATP
value of 170 µM, a KRib-5-P value
of 110 µM, and a Vmax value of
13.1 µmol (min × mg of protein)
1.

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Fig. 1.
Reaction mechanism of PRPP synthase.
Activity was determined as described in "Experimental Procedures."
The MgCl2 concentration was 5.0 mM.
Lines represent fitting of the data to Equation 2.
A, double reciprocal plot of velocity versus ATP
at five concentrations of Rib-5-P. The concentration of ATP was varied
from 0.05 to 1.0 mM in the presence of different
concentrations of Rib-5-P: 1, 0.125 mM;
2, 0.25 mM; 3, 0.50 mM;
4, 1.00 mM, and 5, 2.00 mM. B, double reciprocal plot of initial
velocity versus Rib-5-P at four concentrations of ATP. The
concentration of Rib-5-P was varied from 0.125 to 2.00 mM
in the presence of different concentrations of ATP: 1, 0.05 mM; 2, 0.10 mM; 3, 0.25 mM; and 4, 1.00 mM.
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The binding order was analyzed by product inhibition studies. Rib-5-P
or ATP was varied at different fixed concentrations of the products
PRPP or AMP. Inhibition by PRPP was competitive with respect to ATP
(Fig. 2A) and noncompetitive
with respect to Rib-5-P (Fig. 2B). Inhibition by AMP was
noncompetitive with respect to both ATP and Rib-5-P (Fig. 2,
C and D). The calculated inhibition constants are
given in Table I. The data agree with a
steady-state ordered Bi Bi mechanism in which ATP binds to the enzyme
first, followed by Rib-5-P and a leaving order of products where AMP
release is followed by release of PRPP.

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Fig. 2.
Product inhibition of PRPP synthase.
Activity was determined as described in "Experimental Procedures."
A, double reciprocal plot of initial velocity
versus ATP at four concentrations of PRPP. The
concentrations of Rib-5-P and MgCl2 were 3.0 and 5.0 mM, respectively. The concentration of ATP was varied from
0.1 to 2.0 mM in the presence of different concentrations
of PRPP: 1, 0.0 mM; 2, 0.5 mM; 3, 1.0 mM; and 4, 2.0 mM. The lines
represent fitting of the dataset to Equation 3. B, double
reciprocal plot of initial velocity versus Rib-5-P at four
concentrations of PRPP. The concentrations of ATP and MgCl2
were 2.0 and 5.0 mM, respectively. The concentration of
Rib-5-P was varied from 0.1 to 1.0 mM in the presence of
different concentrations of PRPP: 1, 0.0 mM;
2, 0.5 mM; 3, 1.0 mM; and
4, 2.0 mM. The lines represent
fitting of the dataset to Equation 4. C, double reciprocal
plot of initial velocity versus ATP at four concentrations
of AMP. The concentrations of Rib-5-P and MgCl2 were 3.0 and 5.0 mM, respectively. The concentration of ATP was
varied from 0.1 to 1.0 mM in the presence of different
concentrations of AMP: 1, 0.0 mM; 2,
0.5 mM; 3, 1.0 mM; and 4,
2.0 mM. The lines represent fitting of the
dataset to Equation 4. D, double reciprocal plot of initial
velocity versus Rib-5-P at four concentrations of AMP. The
concentrations of ATP and MgCl2 were 2.0 and 5.0 mM, respectively. The concentration of Rib-5-P was varied
from 0.1 to 1.0 mM in the presence of different
concentrations of AMP: 1, 0.0 mM; 2,
0.2 mM; 3, 0.4 mM; and
4, 1.0 mM. The lines represent
fitting of the dataset to Equation 4.
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Table I
Inhibition constants and mode of inhibition of PRPP synthase
Inhibition constants were determined as described in "Experimental
Procedures." Standard errors are those given by the computer program.
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Alternate Diphosphoryl Donors--
Spinach PRPP synthase isozyme 3 readily accepted nucleoside triphosphates other than ATP as
diphosphoryl donors. Saturation with dATP, CTP, or UTP followed
Michaelis-Menten kinetics, and data were fitted to Equation 1. With
GTP, substrate inhibition was observed (Fig.
3). The calculated kinetic constants,
Km, Vapp, and
Ki for GTP, are shown in Table
II. The Km values for
dATP, CTP, and UTP varied little from KATP,
whereas the Km value for GTP was 3- to 4-fold
higher. The Vapp values for CTP and UTP were 10% of Vmax (Table II). As a result dATP and
ATP were essentially equally effective as diphosphoryl donors, whereas
GTP, CTP, and UTP were less efficient compared with ATP.

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Fig. 3.
Saturation of PRPP synthase with GTP.
Activity was determined as described in "Experimental Procedures."
The concentration of GTP was varied from 0.125 to 8.0 mM in
the presence of 3.0 mM Rib-5-P and MgCl2 in at
least 2 mM in excess of diphosphoribosyl donor
concentration. v is expressed as µmol (min × mg of
protein) 1. Data were fitted to Equation 5.
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Table II
Kinetic constants of PRPP synthase for diphosphoryl donors other than
ATP
Kinetic constants were determined as described in "Experimental
Procedures." Standard errors are those given by the computer program.
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In contrast to the diphosphoryl donor, the enzyme was strictly specific
for the ribose moiety. Thus, activity of the enzyme with deoxyribose
5-phosphate as substrate was undetectable.
Inhibition by Ribonucleotides--
ADP and in some cases
GDP, are potent inhibitors of PRPP synthases from a variety of
organisms. Both of these ribonucleotides also inhibited spinach PRPP
synthase isozyme 3. However, both ADP (Fig.
4A and Table I) and GDP (data
not shown) were linear competitive inhibitors of ATP saturation.
Furthermore inhibition by ADP was noncompetitive with respect to
Rib-5-P (Fig. 4B and Table I). The inhibitory effect of GTP
on PRPP synthase activity was analyzed further (Fig.
5). The results are interpreted as simple
competitive inhibition of binding of ATP by GTP. The
KiGTP value at a given ATP concentration can be
estimated as [ATP] × KmGTP/KATP. At 1 mM ATP this value is 3.8 mM, whereas at 3 mM ATP the value is 11.5 mM. Both values are
consistent with those extrapolated from the data of Fig. 5. Thus, ATP
relieved the inhibition by GTP. This indicates that the cause of
substrate inhibition by GTP most likely resulted from the formation of
a catalytically noncompetent complex of two or more GTP molecules bound
in the active site.

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Fig. 4.
Inhibition of PRPP synthase activity by
ADP. Activity was determined as described in "Experimental
Procedures." The MgCl2 concentration was 5.0 mM. A, double reciprocal plot of initial
velocity versus ATP at four concentrations of ADP. The
concentration of ATP was varied from 0.2 to 2.0 mM in the
presence of different concentrations of ADP: 1, 0.0 mM; 2, 0.5 mM; 3, 1.0 mM, and 4, 2.0 mM. The
lines represent fitting of the dataset to Equation 3.
B, double reciprocal plot of initial velocity
versus Rib-5-P at four concentrations of ADP. The
concentration of Rib-5-P was varied from 0.1 to 1.0 mM in
the presence of different concentrations of ADP as described in
A. The lines represent fitting of the dataset to
Equation 4.
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Fig. 5.
Inhibition of PRPP synthase activity by
GTP. Activity was determined with [ -32P]ATP as
described in "Experimental Procedures." The concentration of
Rib-5-P was 3 mM, whereas the concentration of
MgCl2 was 2 mM in excess of the total
ribonucleoside triphosphate concentration. The concentration of
unlabeled GTP was varied from 0.0 to 3.0 mM at an ATP
concentration of 5.0 mM ( ), 3.0 mM ( ), or
1.0 mM ( ).
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Neither tryptophan nor histidine (up to 8 mM, each), which
are also products of the PRPP consuming pathways, inhibited the activity of spinach PRPP synthase isozyme 3.
 |
DISCUSSION |
We here show that spinach PRPP synthase isozyme 3 had properties
very different from other well characterized PRPP synthases. These
properties were independence of Pi for activity and
stability, lack of allosteric inhibition, and relaxed specificity for
diphosphoryl donor. The classical PRPP synthases share common
properties, among others, of being dependent on Pi for
maximal activity and being allosterically inhibited by ADP. In fact
evidence has accumulated, which indicate that Pi and ADP
both bind to the same allosteric site. This evidence comes from results
of characterization of six point mutations of human PRPP synthase 1. These mutant variants are associated with gouty arthritis because of
overproduction of purine compounds. The mutant forms are less sensitive
or insensitive to ADP inhibition and simultaneously are activated by
lower concentrations of Pi than the normal enzyme (22). In
addition the crystal structure of B. subtilis PRPP synthase
has been obtained either in complex with
,
-methylene ADP, an
analog of ADP, or with SO42
, an analog of
Pi. In these two complexes the
-phosphorus of
,
-methylene ADP and SO42
occupy the same
position (23). Finally, analysis of ADP inhibition of Pi
activation of E. coli PRPP synthase suggests that the two compounds bind to the same site (24). The present work demonstrates that the activity of spinach PRPP synthase isozyme 3 was independent of
Pi and that the enzyme was inhibited by ADP only in a
manner competitive with ATP. This is in contrast to the complex ADP
inhibition found for the Salmonella typhimurium PRPP
synthase (25). Together these observations suggest that spinach PRPP
synthase isozyme 3 lacks a functional allosteric site for
ribonucleoside diphosphates. GTP also inhibited spinach PRPP synthase
isozyme 3. This effect appeared to be caused by binding of two
molecules of GTP to the active site. Similar observations have been
done with various nucleotides inhibiting PRPP synthase from S. typhimurium (25). From the data in Fig. 5, we conclude that there
seems to be no alternative allosteric binding site to which binding of
GTP will inhibit the enzyme activity. In addition, the classical PRPP
synthases use ATP exclusively as diphosphoryl donor, except for the
mammalian enzymes, which may also use dATP (26, 27). Contrary to this, spinach PRPP synthase isozyme 3 had a much broader specificity toward
diphosphoryl donor by using, in addition to ATP or dATP, GTP or the
pyrimidine ribonucleoside triphosphates CTP or UTP.
The amino acid sequence identity of spinach PRPP synthase isozyme 3 with the classical PRPP synthases from E. coli, S. typhimurium, B. subtilis, and mammalian sources is low.
Thus, the identity of spinach PRPP synthase isozyme 3 with E. coli and B. subtilis PRPP synthases and human PRPP
synthase 1 is 23-25%, whereas the identity of E. coli PRPP
synthase with B. subtilis PRPP synthase and human PRPP
synthase 1 is 52 and 49%, respectively. Based on amino acid sequence
comparison we previously suggested a distinction between Class I
(i.e. classical) PRPP synthases and Class II PRPP synthases
(6). This low sequence identity of spinach PRPP synthase isozyme 3 with
the classical PRPP synthases is reflected in the dramatic differences
in enzymatic properties between spinach PRPP synthase isozyme 3 and the
classical PRPP synthases reported in the present work. Class II appears
to be specific for plants and contains a few other members: spinach
PRPP synthase isozyme 4, A. thaliana PRPP synthase isozymes
3 and 4. These PRPP synthases have been shown to be independent of
Pi for activity (6, 7). Six additional partial sequences
with high homology to spinach PRPP synthase isozyme 3 have been
identified by database search. All of these sequences originate from
plant sources (GenBankTM/EBI accession no. AI973393
(maize), AW620825 (soybean), AW696707 (barrel medic), BE041040 (rice),
BE341210 (potato), and H75122 (loblolly pine)). Spinach and A. thaliana contain at least two and three additional PRPP synthases,
respectively. Enzymatic analysis or amino acid sequence comparison
showed that these enzymes belong to Class I (6, 7).
Spinach PRPP synthase isozyme 3 also shares common properties with the
classical PRPP synthases. The kinetic mechanism of spinach PRPP
synthase isozyme 3 was steady-state ordered Bi Bi with MgATP binding
first and PRPP leaving last similar to that shown previously for
S. typhimurium and E. coli PRPP synthases (21,
28). In addition, spinach PRPP synthase isozyme 3 required free
Mg2+ as an activator, similar to what has been shown for
the bacterial and human PRPP synthases (8, 21, 26, 29).
Previous reports on plant PRPP synthases describe some properties of an
enzyme purified 14-fold from spinach leaves (30) or an enzyme purified
from rubber tree latex (31). The latter enzyme apparently is different
from all other PRPP synthases in size and specific activity. The
molecular mass of the subunit is estimated as 57,000 Da, compared with
35,497 Da of spinach PRPP synthase isozyme 3. The activity of the
purified rubber tree latex enzyme is 0.7 µmol (min × mg of
protein)
1, which is much lower than the 13 and 170 µmol
(min × mg of protein)
1 determined for the spinach
PRPP synthase isozyme 3 and E. coli PRPP synthase (28),
respectively. It is very likely that these preparations of PRPP
synthases from spinach leaves and from rubber tree latex are composed
of more than one subunit species. In contrast, we used a highly
purified enzyme specified by an allele of the spinach PRS3
cDNA in the present work.
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ACKNOWLEDGEMENTS |
We thank Robert L. Switzer for careful
reading of the manuscript, Martin Willemoës for invaluable
discussions and for careful reading of the manuscript, and Tonny D. Hansen for excellent technical assistance.
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FOOTNOTES |
*
This work was supported by the Center for Enzyme Research
and the Danish Natural Science Research Council.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. Tel.: 45 3532 2027;
Fax: 45 3532 2040; E-mail: hove@mermaid.molbio.ku.dk.
Published, JBC Papers in Press, February 27, 2001, DOI 10.1074/jbc.M010172200
2
H. Bujard, University of Heidelberg, personal communication.
 |
ABBREVIATIONS |
The abbreviations used are:
PRPP, 5-phospho-D-ribosyl
-1-diphosphate;
Rib-5-P, ribose
5-phosphate.
 |
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