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(Received for publication, November
3, 1995; and in revised form, February 27, 1996) From the
Human UMP synthase is a bifunctional protein containing two
separate catalytic domains, orotate phosphoribosyltransferase (EC
2.4.2.10) and orotidine-5`-phosphate decarboxylase (EC 4.1.1.23). These
studies address the question of why the last two reactions in
pyrimidine nucleotide synthesis are catalyzed by a bifunctional enzyme
in mammalian cells, but by two separate enzymes in microorganisms. From
existing data on subunit associations of the respective enzymes and
calculations showing the molar concentration of enzyme to be far lower
in mammalian cells than in microorganisms, we hypothesize that the
covalent union in UMP synthase stabilizes the domains containing the
respective catalytic centers. Evidence supporting this hypothesis comes
from studies of stability of enzyme activity in vitro, at
physiological concentrations, of UMP synthase, the two isolated
catalytic domains prepared by site-directed mutagenesis of UMP
synthase, and the yeast ODCase. The two engineered domains have
activities very similar to the native UMP synthase, but unlike the
bifunctional protein, the domains are quite unstable under conditions
promoting the dissociated monomer. The final two steps in the de novo biosynthesis of UMP
require the addition of ribose-P to the pyrimidine base orotate by
orotate phosphoribosyltransferase (OPRTase) (
Figure 1:
Genes and possible structures for UMP
synthase. A, natural gene fusion led to the fusion of
catalytic domains. B, conformational states: phosphate and
anionic analogs promote association of inactive subunits to the simple
dimer, as measured by sedimentation. OMP, or nucleotide analogs,
produce an additional conformational change, measured by increased
sedimentation and necessary for optimum enzyme activity.
Monomeric UMP synthase can be converted to a simple dimer, with an s Three different
important benefits are likely candidates for the evolution of the
bifunctional architecture: 1) the two catalytic centers may interact to
``channel'' the intermediate OMP; 2) an element of allosteric
control is jointly communicated between the two different domains; 3)
the intact bifunctional protein is more stable than the separated
catalytic domains. Channeling of intermediate metabolites between
catalytic centers remains a controversial topic as shown in a recent
symposium(7) , with channeling being supported with some
enzymes but not for others. Our earlier studies had tested the
channeling hypothesis for UMP synthase with mouse cell extracts, which
would contain normal quantities of enzymes that might compete for any
OMP being formed by the OPRTase domain of UMP synthase(8) .
These studies showed that channeling was not efficient, since part of
the OMP made by UMP synthase was readily converted to orotidine by an
available phosphatase activity, thereby verifying that some OMP had to
have diffused into the bulk solvent during the assay. There is
better support for the other two benefits, since the dimerization of
UMP synthase in response to effectors results in better activity for
each catalytic domain as suggested in Fig. 1(2, 3, 5, 9) . Also,
such an architecture could serve to enhance the stability of the enzyme
as a monomer or as a dimer. This latter benefit was suggested by the
observation described in the text that the intracellular concentrations
of monofunctional OPRTase and ODCase in microorganisms are
10-100-fold the concentration of the bifunctional UMP synthase in
mammalian cells and was verified by the experiments described.
Plasmid pAcODC was constructed by making a polymerase chain
reaction copy of the ODCase coding region beginning at isoleucine 218.
A primer was designed to change isoleucine to methionine while
incorporating the methionine codon into a new NcoI site. The
polymerase chain reaction product was trimmed with NcoI and
ligated into NcoI cut pAcUMPS. Plasmids of the correct
sequence were identified by restriction analysis and confirmed by
sequencing the DNA across the N-terminal junction. Recombinant plasmids
were grown in Escherichia coli and purified by CsCl gradient
centrifugation in preparation for the production of recombinant
baculoviruses. Thereafter, cabbage looper larvae were injected with
recombinant baculovirus as reported previously(11) . After
injection into the larvae, the expression of the proteins was followed
by Western blots and activity assays with larval extracts, and optimum
expression occurred at 5 days post-infection.
Enzyme activity for
the Arrhenius plots was measured spectrophotometrically, with the
temperature controlled by a thermo-electric cuvette holder. Enzyme was
added at a concentration of 10-15 µg/ml, and substrate
concentrations were at 100 µM orotate plus 0.3 mM P-Rib-PP for the forward OPRTase reaction or at 100 µM OMP plus 1.2 mM PP
where SA is the specific activity of pure enzyme, and the
cytoplasmic volume of tissue is approximately 0.8 ml/g for three
different mammalian tissues(16, 17, 18) .
The human ODCase has a K
The ODCase activity of
the bifunctional UMP synthase at this low protein concentration remains
constant for 40 min at 25 °C (Fig. 2A), and at 37
°C declines very slightly after about 15 min when more than 10% of
the substrate has been depleted. At this low concentration of enzyme,
the ODCase domain is much less stable, and the progress curve becomes
nonlinear after only 2 min at either 25 °C or at 37 °C, before
any significant depletion of substrate has occurred (Fig. 2B). While the ODCase domain and the bifunctional
ODCase have inherently similar activities under optimal conditions
where the enzymes are dimeric, in the experiment of Fig. 2it is
readily apparent that when the ODCase domain is diluted enough to keep
the enzyme partly monomeric, its intrinsic activity is the same as the
activity in UMP synthase for about the first 2 min, whereafter it
becomes much lower than that of the UMP synthase.
Figure 2:
Stability of the dilute human ODCase
catalytic center. Enzyme at a final concentration of 10.5 pM was incubated for the time shown with the substrate OMP at 230
nM, at 37 °C (closed symbols) or at 25 °C (open symbols).
The yeast ODCase
is even more unstable than the human ODCase domain, and activity
declined after only 1 min, and the decline in activity at 37 °C was
more dramatic (Fig. 3). Also, the yeast ODCase showed much lower
total activity at 37 °C, consistent with increased protein
instability at this temperature.
Figure 3:
Stability of dilute yeast ODCase. Enzyme
at a final concentration of 10.1 pM was incubated with the
substrate OMP at 700 nM, at 37 °C (
Similar stability experiments were
done with the OPRTase reaction, but with protein at a concentration of
100 pM to ensure reproducible measurements. For the OPRTase in
UMP synthase, enzyme activity remained linear and stable at both
temperatures and for the full 40-min time period (Fig. 4A). Since our initial studies showed that the
OPRTase domain was a little less active under these experimental
conditions, it was assayed with the domain at a concentration of 1.0
nM. Though at a 10-fold higher concentration than UMP
synthase, the OPRTase domain's activity was not linear at 37
°C or at 25 °C (Fig. 4B). While the diluted
ODCase domain appeared to be equally unstable at 37 °C or at 25
°C (Fig. 2B), the OPRTase domain appeared more
unstable at the lower temperature (Fig. 4B).
Figure 4:
Stability of the dilute human OPRTase
catalytic center. Enzyme at a final concentration of 100 pM (A) or at 1.0 nM (B) was incubated for
the time shown with the substrate orotate at 2.0 µM, at 37
°C (closed symbols) or at 25 °C (open
symbols).
To test
these activities at physiological concentrations of enzyme, an effort
was made to estimate what these concentrations might be, by using data
from published purification results (see ``Experimental
Procedures''). It is apparent that in four normal mammalian
tissues, the bifunctional UMP synthase has cellular concentrations
estimated to be in the 11-32 nM range (Table 2).
By comparison, mitotically active tumor cells have increased the
concentration of this important bifunctional protein by about 10-fold
or more(20, 21) , while microorganisms have
concentrations of the separate catalytic enzymes that are very much
higher (Table 2). Therefore, additional stability experiments
were done with enzyme concentrations at 40 nM, a value at the
upper end of normal physiological concentrations in mammals. The
stability experiment at these higher enzyme concentrations had to be
modified, since activity cannot be maintained for such long time
periods (40 min) at low substrate concentrations. Therefore, enzyme at
a final concentration of about 40 nM was preincubated in
buffer for either one minute, or for 40 min, at which time substrate
was added to a concentration
Figure 5:
Stability of concentrated ODCase at 25
°C. Enzyme at a final concentration of 40 nM was initially
preincubated for 1 min using UMP synthase (-) or ODCase
domain (- - - -), or for 40 min. with UMP
synthase (- - - - -) or ODCase
domain(- - - -). After the preincubation
period, enzyme activity was then initiated by the addition at 0 min of
the substrate OMP at 200 µM.
The OPRTase
domain, by comparison, was more unstable even at these higher
concentrations (40 nM). The 1-min preincubation had no
dramatic effect on the activity, and the curvature evident in Fig. 6reflects the sensitivity of this assay to the accumulation
of products and the possibility of the back reaction. However, when
these same enzyme samples had been preincubated for 40 min, the
bifunctional protein showed a modest decrease in the initial rate of
activity, while the separate OPRTase domain had no detectable activity
when maintained at 25 °C for 40 min in the absence of any ligands.
This dramatic loss of activity was very reproducible. Separate
experiments (not shown) gave no evidence for proteolysis producing the
total loss of enzyme activity seen for the OPRTase in Fig. 6.
Figure 6:
Stability of concentrated OPRTase at 25
°C. Enzyme at a final concentration of 40 nM was initially
preincubated for 1 min using UMP synthase (-) or OPRTase
domain (- - - -) or for 40 min with UMP
synthase (- - - -) or OPRTase domain(-
- - -). Activity was then initiated by the addition
at 0 min with the substrate orotate at 100 µM and P-Rib-PP
at 300 µM.
Although earlier studies with UMP synthase had noted the instability
of the OPRTase activity, this difficulty was resolved by optimizing the
storage and assay buffers as recently described(11) . This
improved stability of the OPRTase activity in the bifunctional native
protein is now evident in Fig. 4A. In the
experiments of Fig. 2Fig. 3Fig. 4, enzyme was used
at a very low concentration to ensure that the protein would be
monomeric in the absence of ligands, and the initial concentration of
the substrate, OMP or orotate, was near K It
is clear that the independent forms of OPRTase and ODCase in bacteria
and yeast must function successfully, so that the instability observed
here was more pronounced due to the experimental conditions. However,
the lower cellular concentration for the mammalian UMP synthase (Table 2) is not a simple benefit of UMP synthase having more
efficient catalytic centers. The opposite is true, since both OPRTases
and ODCases from microorganisms have intrinsic activities that may be
up to 6 or 8 times as fast as the UMP synthase values in Table 1.
Calculated values for k In addition to
the present studies demonstrating that stability is a benefit of the
bifunctional architecture, the extent to which OMP channeling may occur
remains to be explored. While our earlier studies with cell extracts
showed that channeling of OMP was incomplete(8) , with the
availability of pure UMP synthase and the two domains, future studies
will compare the efficiency of OMP transfer within the bifunctional
enzyme in comparison to a mixture of the two domains.
Volume 271,
Number 18,
Issue of May 3, 1996 pp. 10704-10708
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
)to form OMP
and the subsequent decarboxylation of OMP to form UMP by orotidylate
decarboxylase (ODCase). In all microorganisms examined, these two
catalytic centers are coded by two separate genes, while in all
multicellular eukaryotes examined, the genes for these two catalytic
centers have been joined into a single gene, resulting in the
expression of the bifunctional protein, UMP synthase, with two
different catalytic domains (1) (Fig. 1A).
=
inactive; 
= partly active; * =
active.
of 5.1, by various anions
such as phosphate, and to a faster sedimenting 5.6 S dimer with optimal
activity by the normal ligand, OMP, or by nucleotide
analogs(2, 3) (Fig. 1B). An important
feature of this model is that there are two conformational states of
the dimer, based on sedimentation studies showing that only nucleotides
produced the more rapidly sedimenting 5.6 S
species(2, 3) , on studies showing that a different
type of tryptic digestion pattern for the pure enzyme was obtained in
the presence of these tight binding nucleotides, than in the presence
of simple anions(4) , and on studies showing that enzyme
preincubated to be in the 5.6 S form had optimum activity immediately
after the addition of substrate, while enzyme preincubated in the 5.1 S
form had a 20-s lag time after substrate addition before attaining
optimum activity(5) . Such conformational features have been
found for a large set of dissociating enzymes, whose diagnostic feature
is the reversible interconversion between monomeric and oligomeric
forms, which occurs in response to physiological concentrations of
appropriate regulatory effectors(6) .
Materials
The following materials were obtained
from the indicated sources: sodium [7-
C]orotate
and sodium [7-C]OMP (DuPont NEN); polyethylene
glycol, molecular weight 10,000 (Fluka); dithiothreitol, ultrapure
(Boehringer Mannheim); 98-99% pure bovine serum albumin (Sigma).
The yeast ODCase used was either the 60-90% ammonium sulfate
fraction (for the radioactive assays) or the pure enzyme (for the
spectrophotometric assays) produced using the method of Bell and
Jones(10) . All other chemicals were reagent grade or as
specified in the appropriate references.Construction and Expression of Plasmids
Plasmid
pAcUMPS containing the coding region for UMP synthase (11) was
used as a polymerase chain reaction template for construction of
plasmids. With the OPRTase domain at the 5`-end, the plasmid was cut at
a unique NheI site 57 codons into the downstream region of the
ODCase domain. These extra codons of the ODCase domain were removed by
a loop-out mutagenesis using a primer which placed the stop codon
immediately after the codon for glycine 213 of the OPRTase domain in
plasmid pAcOPRT. Mutagenesis was according to the method of Kunkel (12) utilizing the single-stranded DNA produced by phage
M13m19.Purification of Expressed UMP Synthase and the Two
Domains
The expressed UMP synthase, K314C variant, and ODCase
domain were purified on a monoclonal antibody affinity
column(11) . Homogenates of cabbage looper larvae producing
each protein were made using the same protocol except that the larvae
were homogenized in the standard buffer, adjusted to pH 6.8 (at 0
°C) and containing 25% ammonium sulfate. The ODCase domain and
OPRTase domain were precipitated at 65% ammonium sulfate saturation.
After storage of the ammonium sulfate pellet, OPRTase was further
purified by specific elution from a phosphocellulose column. The sample
was diluted 25-fold in buffer (20 mM MOPS, 1 mM EDTA,
pH 6.8) and loaded onto the column (10 ml). The column was then washed
with 100 ml of buffer, 50 ml of buffer containing 0.05 M KCl
followed by 50 ml of buffer, and the last two washes were repeated
until the absorbance at 280 nm was unchanged. The OPRTase domain was
then eluted with 50 ml of buffer containing 0.04 M KCl and 0.1
mM OMP. Elution fractions were concentrated in an Amicon
ultrafiltration cell to a volume of about 8 ml, then in a collodion bag
for about 5 h versus collodion dialysis buffer 2(11) .
Protein concentrations were determined by the method of Bradford (13) .Enzyme Assays
ODCase activity was determined at 25
or 37 °C by measuring the release of CO
from [C]OMP as described previously (14) or by a spectrophotometric assay measuring the decrease in
absorbance at 285 nm for the conversion of OMP to UMP(11) .
OPRTase activity was determined at 25 or 37 °C by measuring the
release of CO from
[C]orotate after sequential conversion to OMP
and UMP + CO or by a spectrophotometric
assay measuring the decrease in absorbance at 295 nm for the conversion
of orotate to OMP and to UMP(11) . Changes to these standard
protocols are given under Results or in figure legends. For assays of
the OPRTase domain in the forward reaction, an equimolar amount of the
human ODCase domain was added to ensure conversion of OMP to UMP +
CO. The specific activities of the
[C]OMP or [C]orotate in
the reaction mixtures were determined by adding large excesses of UMP
synthase to allow total conversion of the substrate. Enzyme dilutions
were made prior to use in the standard buffer.
for the reverse OPRTase
activity or at 200 µM OMP for ODCase activity.Sucrose Density Gradient
Centrifugation
Experiments were generally performed as described
previously(15) . Gradients (10 ml of a 10-40% sucrose
solution containing 10% glycerol) were made in standard buffer: 50
mM Tris-HCl, pH 7.5, 2 mM EDTA, and 5 mM dithiothreitol. Gradients were layered with a 200-µl enzyme
sample containing enzyme protein, plus M
standards. Many gradients also contained effector ligands at
specified concentrations, based on the previously determined affinity
of such ligands for the enzyme.Calculations
Estimates of the concentration in
vivo of the bifunctional UMP synthase or of the monofunctional
ODCase and OPRTase were obtained from published purifications, to
provide the protein concentration, c (nM),
Preparing the Separate Domains
The current data
base shows the consensus sequence for 16 monofunctional OPRTases from
bacteria and fungi does not extend beyond residue 208 of UMP synthase
(480 amino acids total), while a sequence alignment with 31
monofunctional ODCases suggests that the consenus structure for the
ODCase domain begins after amino acid 224. To ensure producing complete
domains, we engineered a plasmid with the sequence for the human UMP
synthase to produce truncated cDNAs for the OPRTase domain ending at
residue 214 and for the ODCase domain beginning at residue 218.
Recombinant baculoviruses for the complete UMP synthase and for both
the OPRTase and ODCase domain expressed proteins of the expected size,
and these domains were specifically recognized in a Western blot by a
polyclonal antibody raised against human UMP synthase (data not shown). Activities
When these protein domains were
purified, they showed enzymatic activity very similar to that observed
with the bifunctional protein. These are the first studies that have
determined both the absolute rates and the K
values for each catalytic center with the bifunctional UMP
synthase, as well as with the separated domains (Table 1). For
the OPRTase, the K for orotate with the
bifunctional human UMP synthase is 2.1 µM, a value almost
the same as measured with the same enzyme from mouse(3) . The
OPRTase domain has a somewhat higher K of 7.1
µM.
of
230 nM for OMP, a value very similar to earlier measurements
for the mammalian enzyme(5, 19) . By comparison the
human ODCase domain had a slightly higher K of 295
nM. These results verify that the domains, produced by our
changes of the human UMP synthase cDNA, behave as if they are properly
folded proteins with normal enzymatic rates and affinities for
substrates.Dimerization of UMP Synthase
Since earlier studies
with the mammalian UMP synthase and the yeast ODCase had demonstrated
the allosteric response of these enzymes to effector ligands as
measured by the association of monomers to form the
dimer(2, 3, 10, 15) , sedimentation
studies were done with the pure human UMP synthase. This conformational
response to allosteric ligands was readily measured with the pure
enzyme: UMP synthase at concentrations below 15 nM was very
stable as a monomer, and it generally took enzyme concentrations 
100 nM to produce association to the dimer in the absence of
an effector nucleotide ligand (data not shown). However, at enzyme
concentrations of about 10 nM, the presence of various high
affinity nucleotides produced the conformational change that stabilized
the dimer form. The tight binding inhibitor nucleotide barbiturate
riboside monophosphate (K = 4.1
nM) produced complete dimerization at a concentration equal to
the enzyme.Dependence of Catalytic Rates on Temperature
The
effect of temperature on OPRTase and ODCase catalytic activities was
tested with enzyme at concentrations of 230-640 nM and
substrates at 100 µM. These optimal concentrations are far
above the physiological concentrations for the enzyme (Table 2),
or the K values determined for their substrates (Table 1). The measured enthalpy was 
H = 9.7
kcal/mol for the forward OPRTase reaction (orotate 
OMP) when
measured with UMP synthase, or with the separated OPRTase domain, and
H = 21.7 kcal/mol for the reverse OPRTase reaction
(OMP orotate). This difference in the intrinsic energy for this
reaction, depending on direction, is completely consistent with earlier
results showing that the reverse reaction is kinetically more favorable
than the forward reaction(3) . For the ODCase reaction
H = 14.9 kcal/mol when measured with UMP synthase
or with the separated ODCase domain. These temperature studies for both
reactions also demonstrate that under optimized experimental conditions
there is no difference in the observed activity for the reaction as
measured with the bifunctional UMP synthase and the separate OPRTase or
ODCase domain, signifying that under these conditions the isolated
proteins are stable and completely active at the temperatures used, and
for the times used.
Thermal Stability of the Two Catalytic
Centers
Since the above experiments were done under conditions
that are clearly not completely physiological, additional studies were
done to properly assess the bifunctional protein and the two separate
domains under conditions that could explore their stability as a
function of protein concentration, which influences whether the enzyme
is a monomer or a dimer. ODCase activity could be reliably measured at
about 10 pM. At this low concentration of enzyme, it was also
possible to use an initial substrate concentration near K, so that the enzyme would not be saturated with
bound substrate and thus increase the detection of instability since
part of the enzyme population would remain unbound to ligand and
therefore in the monomeric form at all times (Fig. 1). To
compare the different progress curves, data are therefore presented as
the percent substrate converted to product.
) or at 25 °C
(
).
50 
K,
and continuous activity was monitored after the addition of substrate
at 0 min to the preincubated enzyme (Fig. 5). When either UMP
synthase or the human ODCase domain was preincubated for 1 min, there
was no detectable change in enzyme activity: the progress curves were
linear until more than 30% of the substrate had been depleted. However,
when the enzyme samples had been preincubated at 25 °C for 40 min
in the absence of any ligands, there was about a 20% decline in the
rate of the initial progress curve, and this decline was comparable for
the bifunctional UMP synthase and for the ODCase domain.
so that
a significant fraction of the enzyme population would remain
ligand-free at any time and therefore be in the monomeric state. Under
these conditions, as a function of time in the assay, enzyme molecules
should alternate between the monomer and active dimer states (Fig. 1). As shown by the results of Fig. 2B and Fig. 3, the isolated human ODCase domain, as well as the yeast
ODCase monofunctional protein, were very unstable under conditions that
favored the monomeric state. Fig. 4B shows a comparable
instability for the OPRTase domain, at a concentration where it would
be expected to be monomeric for part of the assay time. Thus, the data
of Fig. 2A and Fig. 4A are remarkable
in demonstrating the complete stability of each catalytic center when
it is in the bifunctional architecture, even when UMP synthase was
partly in the monomeric form. A possible explanation for the evident
stability of the ODCase and OPRTase centers of the bifunctional UMP
synthase is that there may be interaction between the two different
domains within the same subunit, as modeled in Fig. 1. Even if
such interaction between the OPRTase and ODCase domains is transient,
it could add sufficient stabilization to maintain the active structural
conformation of each domain during the time that the protein subunit is
monomeric, so that there is no measurable loss of activity. By
comparison, both the separate human ODCase domain, the yeast ODCase,
and the separate human OPRTase domain were unstable at concentrations
expected to favor the monomeric state, as measured by dramatic loss of
activity in only a few minutes under otherwise benign conditions.
of OPRTase from bacteria
or yeast are 12-32
s
(22, 23, 24, 25) ,
and the values for ODCase from microorganisms are 37-108
s(10, 26) . Our studies show that
UMP synthase becomes a dimer at an enzyme concentration 100
nM, while at a concentration of 750 nM yeast ODCase
remains monomeric (data not shown). Thus, even though they are
catalytically more efficient, the much larger cellular concentration
for these enzymes in microorganisms may in part be necessary for
stability. At such cellular concentrations, the monofunctioal OPRTase
and ODCase enzymes would be predominantly or totally in the more stable
and active dimeric conformation. By comparison, the bifunctional UMP
synthase does not need to be dimeric for stability, since interaction
between domains within a subunit may serve a similar function, such
that both catalytic centers of UMP synthase remain stable under
conditions where the separated domains lose activity.
)
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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