(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) ()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).
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. =
inactive;
= partly active; * =
active.
Monomeric UMP synthase can be converted to a simple dimer, with an s 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) .
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 for the reverse OPRTase
activity or at 200 µM OMP for ODCase activity.
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 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.
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 () or at 25 °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 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.
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 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.
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 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.
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.