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INTRODUCTION |
Hyperthermophilic organisms exhibit a series of extraordinary
structural and functional adaptations to extreme environmental conditions, in particular to high temperature (1-3). Pyrococcus abyssi, a hyperthermophilic and barophilic/barotolerant archaea belonging to the sulfur metabolizing group (4), grows anaerobically at
temperatures between 67 and 102 °C, with an optimum of 96 °C. Survival of these organisms at these extreme temperatures requires not
only proteins of remarkable thermostability, but also some mechanisms
to protect labile intermediates from thermal degradation.
As in most eukaryotic and prokaryotic organisms, the first two
reactions of the de novo pyrimidine biosynthetic pathway are catalyzed by carbamoyl-phosphate synthetase (CPSase, EC
6.3.5.5)1 and aspartate
transcarbamoylase (ATCase, EC 2.1.3.2). The product of the CPSase
reaction, carbamoyl phosphate, is also a substrate for ornithine
transcarbamoylase (OTCase, EC 2.1.3.3) in the arginine biosynthetic
pathway. P. abyssi contains a single CPSase (5), which
provides carbamoyl phosphate for both pyrimidine and arginine
biosynthesis. This intermediate is extremely unstable at elevated
temperature. At 96 °C, the optimum growth temperature of P. abyssi, the half-life of carbamoyl phosphate is only 2-3 s (6,
7). Consequently, protective mechanisms are likely to be operative
ensuring that carbamoyl phosphate is efficiently delivered to the next
enzyme in the reaction sequence without significant thermal degradation.
Metabolic channeling, a process whereby the product of one enzyme is
directly transferred to the next enzyme in the pathway without being
released into the bulk solvent, provides a means of sequestering
unstable intermediates. The transfer of intermediates requires physical
association of the coupled enzymes either as stable complexes such as
E. coli tryptophan synthetase (8, 9) or by transient contact
as proposed for glycolytic enzymes (10). Partial channeling of
carbamoyl phosphate has been reported in the pyrimidine biosynthetic
complexes from yeast (11-13), Neurospora (14, 15), and
mammals (16-21) and in the mammalian urea cycle enzymes (22, 23).
The first two enzymes of the de novo pyrimidine biosynthetic
pathway of P. abyssi were recently isolated and
characterized (5, 24, 25). The subunit structure of P. abyssi ATCase resembles Escherichia coli ATCase, in a
sense that it is comprised of 34-kDa catalytic and 17-kDa regulatory
subunits. Remarkably, CPSase activity in P. abyssi is
associated with a 35-kDa polypeptide and is thus much smaller than the
120-kDa domain or subunit which catalyzes this reaction in mesophilic
organisms. The insensitivity of ATCase to carbamoyl phosphate analogs,
phosphonacetate and pyrophosphate (24), suggested that the carbamoyl
phosphate-binding site is shielded to some extent from the bulk solvent
and that the intermediate may be sequestered within a complex.
In this report, isotope dilution experiments and pre-steady state
kinetic measurements (26) have directly demonstrated carbamoyl phosphate channeling between P. abyssi CPSase and both
ATCase and OTCase. At ambient temperatures, the channeling is leaky, but the channeling efficiency appreciably increases at elevated temperature.
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EXPERIMENTAL PROCEDURES |
Chemicals--
[14C]Sodium bicarbonate (57.3 mCi/mmol) was obtained from NEN Life Science Products Inc.;
[U-14C]aspartate (300 mCi/mmol) and
[14C]ornithine (210 mCi/mmol) from CEA-Saclay, Service of
Biochemistry; aspartate, carbamoyl phosphate, ornithine,
trichloroacetic acid, and hydroxylamine were from Sigma; Sephacryl S300
Superfine was from Pharmacia. Bio-Rex 70 from Bio-Rad;
N-(phosphonacetyl)-L-aspartate (PALA) was a gift
from Drs. V. Narayanan and L. Kedda of the Drug Synthesis and Chemistry
Branch, Division of Cancer Treatment, National Institutes of Health,
Bethesda, MD.
Strains and Cell Growth--
P. abyssi strain GE5 was
isolated and characterized by Erauso et al. (4). These cells
were grown at 95 °C under anaerobic conditions in enriched
artificial seawater containing elemental sulfur (4, 5). E. coli EK1104 (F
ara thi
pro-lac
pyrB pyrF±
rpsL) (27). The plasmid pKSAT3, which encodes the P. abyssi pyrBI operon in Bluescript II KS+ vector (25), was
transformed into E. coli strain EK1104 for expression of the
recombinant P. abyssi ATCase.
Enzyme Preparations--
P. abyssi and E. coli cell-free extracts were prepared by resuspending the frozen
cells in buffer A (50 mM Tris-HCl, pH 8, 1 mM
dithiothreitol, and 0.1 mM EDTA) and sonication six times for 30 s using a Biosonik III sonicator at 20 kilocycles/s. The extracts were dialyzed against the same buffer overnight at
4 °C.
P. abyssi CPSase was purified as described previously (5).
Due to the instability of the protein during later stages of purification, the procedure was carried through ion-exchange and size
exclusion chromatography steps (28) resulting in a 43-fold purification
of the enzyme. E. coli ATCase was isolated from EK1104 cells
transformed with the plasmid pEK2 (27).
Enzymes Assays--
Carbamoyl-phosphate synthetase activity was
measured using the method described by Abdelal and Ingraham (29), which
relies on the conversion of [14C]carbamoyl phosphate to
hydroxylurea. The 0.300-ml assay mixture (25) was incubated at
37 °C for 15 min, quenched with 30 µl of 1.2 M
hydroxylamine, and then incubated for 10 min at 100 °C. After
addition of 0.7 ml of 10% trichloroacetic acid, the samples were
treated as described below for the coupled reaction.
Aspartate transcarbamoylase activity was assayed by a procedure (30) in
which radiolabeled carbamoyl aspartate synthesized from
[U-14C]aspartate is isolated by ion exchange
chromatography. The substrate concentrations in the 0.300-ml assay
mixture and the incubation time were adapted to each temperature as
described previously (24).
Ornithine transcarbamoylase activity was assayed by measuring the
formation of radiolabeled citrulline from [14C]ornithine
(31) or [14C]carbamoyl phosphate (32). The assay mixture,
consisting of 0.2 mM [14C]ornithine (200,000 cpm/µmol), 5 mM carbamoyl phosphate, and 50 mM Tris-HCl, pH 8, was incubated at 37 °C for 10 min in
a final volume of 1.0 ml and then quenched by addition of 100 µl of 1 N HCl. The product, [14C]citrulline, was
isolated by chromatography on Bio-Rex 70 cation exchange column when
the substrate was [14C]ornithine or by removing unreacted
radiolabeled [14C]carbamoyl phosphate by quenching in
10% trichloroacetic acid and heating at 100 oC (32).
The progress curve for the CPSase-ATCase-coupled reaction was obtained
by measuring the time-dependent formation of
[14C]carbamoyl aspartate. The assay mixture consisted of
60 mM [14C]bicarbonate (200,000 cpm/µmol),
120 mM NH4Cl, 0.75 mM ATP, and 20 mM aspartate, 50 mM Tris-HCl, pH 8, in a total
volume of 300 µl (5, 32). Assays were conducted at 37, 70, and
90 °C and the concentrations of all the substrates was varied as
described previously (5). The same procedure was used to measure the CPSase-OTCase-coupled reaction, except that aspartate was replaced with
0.2 mM ornithine. The steady state rate of carbamoyl
aspartate or citrulline formation, Vo, was
determined by a least squares fit of the data to the Easterby equation
(34). The transient time (
) was then obtained by extrapolating the
steady state rate to the time axis. Continued extrapolation to the
concentration axis gave
[I], where [I] is
the steady state concentration of the carbamoyl phosphate. Because
[I] is dependent on Vo, the observed
values were normalized to the velocity obtained for the progress curve
at 37 °C in cell extracts, using the expression,
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(Eq. 1)
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One unit of CPSase, ATCase, or OTCase activity is defined as the
formation of 1 µmol of product per hour. Specific enzymatic activity
is expressed as units/mg of enzyme or, in the case of cell-free
extracts, of total protein. Protein concentration was determined by the
Lowry method (33), using bovine serum albumin as standard. Substrate
saturation curves were fit to the Michaelis-Menten equation by least
square analysis using the program KaleidaGraph.
Channeling was analyzed using the approach devised by Ovádi
et al. (26) for coupled enzymes, E1
and E2, that catalyze sequential reactions. The
steady state velocity of the individual reactions catalyzed by each of
these enzymes, VE1 and
VE2, was measured using their respective
substrates.
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(Eq. 2)
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The overall reaction rate of the coupled enzyme system,
Vo, was also measured as a function of time in the
presence of all substrates. For partial channeling, the following
criteria must be satisfied,
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(Eq. 3)
|
where k
2(1) and
k
2 are the pseudo first-order rate constants
for the second reaction in the presence and absence of the first enzyme
E1, respectively.
Size Exclusion Chromatography--
To determine whether the
P. abyssi enzymes form complexes that could be isolated,
2-ml aliquots (20 mg) of the dialyzed cell-free extracts were applied
to a 1.4 × 50-cm Sephacryl S-300 Superfine column equilibrated
with 50 mM Tris-HCl, pH 8, 1 mM dithiothreitol, and 0.1 mM EDTA. The column was eluted with the same buffer
at a flow rate of 0.15 ml min-l and fractions (1.0-1.7 ml)
were collected and assayed for CPSase, ATCase, and OTCase
activity. Column temperature was controlled between 23 and 70 °C by
circulating water through the column jacket. Substrates or inhibitors
were added to the dialysis and elution buffers as indicated in the text.
 |
RESULTS |
P. abyssi Ornithine Transcarbamoylase--
The catalytic and
regulatory properties of P. abyssi CPSase and ATCase were
previously described (5, 24). Prior to undertaking the channeling
studies it was necessary to characterize the OTCase activity in
dialyzed P. abyssi cell-free extracts. At 37 °C in the
presence of saturating concentrations of carbamoyl phosphate and
ornithine, the OTCase specific activity was found in six determinations to be 0.42 ± 0.02 units/mg of cell protein. The rate of product formation is linear for at least 15 min.
The carbamoyl phosphate and ornithine saturation curves obtained at
37 °C conformed to Michaelis-Menten kinetics. Nonlinear least
squares analysis of this data gave a Vmax of
0.62 ± 0.08 units/mg, and Km for carbamoyl
phosphate and ornithine of 8.0 ± 1.4 and 28.5 ± 5.1 µM, respectively. It is noteworthy, that for OTCase, the
Km for the carbamoyl phosphate is 7.5-fold lower
than the [S]0.5 value (60.0 ± 2.3 µM) obtained for P. abyssi ATCase (24),
suggesting an appreciably higher affinity for the intermediate.
Isotope Dilution in the CPSase-ATCase and CPSase-OTCase-coupled
Reactions--
Channeling can be detected by measuring the extent to
which [14C]carbamoyl phosphate produced endogenously by
CPSase is diluted by unlabeled carbamoyl phosphate added to the assay
mixture.2
CPSase-ATCase-coupled Reaction--
Dilution of endogenous
carbamoyl phosphate was measured in P. abyssi GE5 dialyzed
cell-free extracts containing constitutive levels of all three enzymes,
CPSase, ATCase, and OTCase. However, the CPSase-ATCase-coupled reaction
could be isolated since the omission of ornithine from the assay
mixture resulted in less than 0.1% incorporation of the carbamoyl
phosphate into citrulline. At 37 °C, in the presence of saturating
[14C]sodium bicarbonate, MgATP, NH4Cl, and
aspartate, the incorporation of radiolabeled substrate into carbamoyl
aspartate (Fig. 1) was unaffected by
unlabeled exogenous carbamoyl phosphate at concentrations up to 125 µM, a value 2-fold greater than the observed
[S]0.5 for the intermediate. The concentration
of CPSase substrates, ATP, bicarbonate, and ammonia remain relatively
unchanged, with only 4.6, 0.03, 0.01% consumed during the course of
the reaction. Moreover, the maximum velocity of the ATCase is such that
only 7.9 nmol of carbamoyl phosphate could be consumed during the
30-min incubation reaction, a value that corresponds to 25.9 µM. Thus, the plateau in the dilution curve is not an
artifact resulting in consumption of both endogenous and exogenous
carbamoyl phosphate and represents instead partial compartmentation of
the intermediate. This interpretation is confirmed by control
experiments that assessed isotopic dilution in an uncoupled system. The
same experiment was conducted in extracts of pKSAT3 E. coli
transformants (25). These cells lack E. coli ATCase, so the
carbamoyl phosphate synthesized from [14C]bicarbonate by
the E. coli CPSase can only be incorporated into carbamoyl
aspartate by the recombinant P. abyssi ATCase. In this case
(Fig. 1), the lowest concentrations of exogenous carbamoyl phosphate
effectively competes with the endogenous intermediate and produces an
appreciable dilution of isotope incorporated into carbamoyl
aspartate.

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Fig. 1.
Dilution of [14C]carbamoyl
phosphate incorporation into carbamoyl aspartate or ornithine by
exogenous unlabeled carbamoyl phosphate. The CPSase-ATCase or
CPSase-OTCase-coupled reactions were initiated at 37 °C by the
addition of [14C]bicarbonate and the incorporation of the
radiolabeled carbamoyl aspartate or ornithine during was measured as
described under "Experimental Procedures." To assess isotope
dilution of the P. abyssi CPSase-ATCase-coupled reaction,
the 0.3-ml reaction mixture consisted of saturating CPSase substrates,
the indicated concentration of unlabeled carbamoyl phosphate, 20 mM aspartate, and 24.7 µg of P. abyssi
dialyzed cell-free extract ( ). The CPSase activity was 0.0106 units,
so that 5.29 nmol of endogenous [14C]carbamoyl phosphate
is incorporated into carbamoyl aspartate during the 30-min incubation
period in the absence of exogenous unlabeled carbamoyl phosphate
(relative amount = 1.0). The ATCase activity was 0.63 units/mg.
For the P. abyssi CPSase-OTCase-coupled reaction ( ), the
same reaction was carried out with 24.7 µg of P. abyssi
extract and 0. 2 mM ornithine. In this experiment, the
CPSase activity was 0.0103 units (5.16 nmol corresponds to a relative
amount = 1.0) and the OTCase activity was 0.465 units/mg. As a
control, P. abyssi CPSase was coupled to E. coli
ATCase by carrying out the same reaction in the presence of 31.5 µg
of pKSAT3 dialyzed cell-free extract and 20 mM aspartate
( ). The CPSase activity was 0.0041 units (2.06 nmol corresponds to a
relative amount = 1.0) and the ATCase activity was 0.42 units/mg.
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In the P. abyssi CPSase-ATCase-coupled reaction, dilution of
the endogenous intermediate by high concentrations of exogenous carbamoyl phosphate demonstrates that under these conditions channeling is partial or leaky, not absolute. Nevertheless, these experiments clearly show that in P. abyssi the endogenously synthesized
intermediate does not freely equilibrate with carbamoyl phosphate in
the bulk phase.
Judging from the altered response to allosteric effectors (24), the
conformational transitions exhibited by P. abyssi ATCase appear to be influenced by temperature. The lability of carbamoyl phosphate makes it impractical to conduct the isotope dilution experiments at elevated temperature. Consequently, the temperature dependence of channeling in the CPSaseATCase-coupled reaction was
investigated by carrying out the same experiment at reduced temperature. The dilution profiles obtained at 37 and 22.5 °C are
similar at higher concentrations of carbamoyl phosphate, but at
22.5 °C, isotope dilution begins to occur at much lower
concentrations of exogenous carbamoyl phosphate (Fig.
2). Nevertheless, it is clear that
channeling still occurs at the lower temperature.

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Fig. 2.
Effect of reduced temperature on carbamoyl
phosphate isotope dilution in the CPSase-ATCase-coupled reaction.
P. abyssi dialyzed cell-free extracts were incubated at
22.5 °C in the presence of saturating concentrations of CPSase
substrates, of 20 mM aspartate and increasing
concentrations of unlabeled carbamoyl phosphate as described in the
legend to Fig. 1 and under "Experimental Procedures." The CPSase
activity was 0.0149 units, so that 7.44 nmol of endogenous
[14C]carbamoyl phosphate is incorporated into carbamoyl
aspartate during the 30-min incubation period in the absence of
exogenous unlabeled carbamoyl phosphate (relative amount = 1.0).
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CPSase-OTCase-coupled Reaction--
The effect of exogenous
carbamoyl phosphate on the incorporation of the radiolabeled
intermediate into citrulline was also measured at 37 °C in the
P. abyssi cell-free extracts employing the same saturating
concentrations of CPSase substrates and 0.2 mM ornithine
(Fig. 1). Aspartate was omitted from the assay mixture, so that the
formation of radiolabeled carbamoyl aspartate was negligible. Again, no
significant isotope dilution occurs up to a concentration of exogenous
carbamoyl phosphate of 300 µM which is 38-fold higher
than the Km for carbamoyl phosphate of the OTCase,
suggesting that the intermediate is also partially channeled to the
arginine biosynthetic enzyme. As in the case of the
CPSase-ATCase-coupled reaction only a small fraction of the CPSase
substrates (4.3% of the ATP) is utilized during the course of the
reaction, and the maximum amount of carbamoyl phosphate that could be
converted to citrulline by OTCase was 5.7 nmol, corresponding to a
concentration of 19.1 µM.
Transient Time Measurements of the Coupled Reactions--
The
transient time (
), a parameter that
reflects3 the time required
for a coupled enzyme system to reach steady-state, and the effective
concentration of intermediate (34, 35) can be obtained from the
progress curve for the formation of the final product (see
"Experimental Procedures"). The parameters for unlinked enzymes can
be directly determined under circumstances where no channeling would be
expected, in this case the coupled reaction between the P. abyssi CPSase and E. coli ATCase. For perfect or absolute channeling both
and [I] are zero.
Intermediate values for these parameters are indicative of partial or
leaky channeling.
CPSase-ATCase-coupled Reaction--
The time course of carbamoyl
aspartate formation for the CPSase-ATCase-coupled reaction was obtained
for the endogenous enzymes in GE5 dialyzed cell-free extracts as well
as purified P. abyssi CPSase in the presence of either
partially purified P. abyssi ATCase or purified E. coli ATCase.
At 37 °C using cell-free extracts, the progress curve for carbamoyl
aspartate formation in the presence of saturating substrates reaches
steady state after a short lag phase and remains constant for at least
40 min. Analysis of this data gave a
of 16 s and a steady
state concentration of carbamoyl phosphate [I] of 0.34 µM (Table I). In contrast,
when P. abyssi CPSase was coupled to E. coli
ATCase at the same temperature, values of 51 s and 5.8 µM were obtained for
and [I],
respectively. The carbamoyl phosphate concentration depends on the
final steady state velocity of carbamoyl aspartate formation, so all
values for intermediate concentration were normalized (see
"Experimental Procedures") to the observed rate at 37 °C
obtained with cell extracts. The normalized value for the steady state
carbamoyl phosphate concentration in the P. abyssi
CPSase-E. coli ATCase-coupled reaction was 2.23 µM or about 7-fold higher than the value obtained for
P. abyssi cell extracts. The shorter transient time and
lower steady state carbamoyl phosphate concentration is consistent with
the idea that the intermediate is partially channeled between the
active sites of the P. abyssi enzymes.
At 70 °C in cell-free extracts, the steady state rate of product
formation remains linear for only 2 min. The transient time was found
to be 3.3 s (Table I), a value that is 5-fold smaller than the
value obtained at 37 °C. The increased velocity at elevated temperature results in an appreciably higher carbamoyl phosphate concentration. However, the normalized steady state concentration of
the intermediate at measured at 70 °C is 0.17 µM.
Similar results were obtained for a mixture of isolated P. abyssi CPSase and ATCase (Fig.
3A). The transient time was
4.0 s and the normalized steady state carbamoyl phosphate
concentration was 0.17 µM, in good agreement with the
values obtained for dialyzed cell-free extracts at the same
temperature. The progress curve measured at 70 °C for the purified
P. abyssi CPSase coupled to E. coli ATCase gives
a transient time of 23.9 s and a normalized steady state
carbamoyl phosphate concentration of 1.04 µM.

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Fig. 3.
Progress curves for the formation of
carbamoyl aspartate in the CPSase-ATCase-coupled reactions. The
time dependent formation of carbamoyl aspartate in the
CPSase-ATCase-coupled reaction was measured as described under
"Experimental Procedures." Panel A, the 0.300-ml
reaction mixture consisted of saturating
[14C]bicarbonate, ATP, NH4Cl, and aspartate,
2 µg of purified P. abyssi CPSase and either 3 µg of
partially purified P. abyssi ATCase ( ) or 37 µg of
E. coli ATCase ( ), at 70 °C. Panel B, the
same reaction was carried out at 90 °C with 2 µg of purified
P. abyssi CPSase and 3 µg of partially purified P. abyssi ATCase.
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Remarkably, at 90 °C both the transient time and steady state
concentration of carbamoyl phosphate for the P. abyssi
CPSase-ATCase homologous couple are immeasurably small (Fig.
3B and Table I) suggesting that at temperatures close to the
optimal growth rate of the organism carbamoyl phosphate channeling is
absolute. The corresponding control using the heterologous enzyme
system could not be carried out at 90 °C since E. coli
ATCase is instantaneously inactivated at this temperature.
CPSase-OTCase-coupled Reaction--
Transient time measurement at
37 °C indicates that carbamoyl phosphate is also partially channeled
to ornithine transcarbamoylase. In these experiments, the progress
curve for citrulline formation was measured using dialyzed extract
supplemented with saturating ornithine and no aspartate. The transient
time of 31.2 s (Table I) and the steady state concentration of
carbamoyl phosphate, when corrected for differences in reaction rate,
of 0.26 µM are comparable to the values obtained for the
CPSase-ATCase-coupled reaction at 37 °C in cell extracts. These
values are appreciably lower than those obtained when P. abyssi CPSase is coupled to E. coli
ATCase.
Analysis of Channeling using the Ovádi
Formalism--
Ovádi and associates (26) have developed a series
of criteria designed to assess channeling in coupled enzyme systems. For this analysis, the rate of the individual reactions catalyzed by
CPSase (VE1) and either ATCase or OTCase
(VE2), as well as the rate of the
coupled reactions, CPSase-ATCase or CPSase-OTCase
(Vo), the reciprocal transient time
(1/
obs), the pseudo first-order rate constant of the
individual reaction catalyzed by E2 alone
(k
2) and in the presence of
E1
(k
2(1)), were measured at 37 and
70 °C in dialyzed extracts. The kinetic parameters, summarized in
Table II, show that both coupled
reactions satisfy the Ovádi criteria for "leaky" or partial
channeling. For the CPSase-ATCase-coupled reaction the
criteria are satisfied at both 37 and 70 °C. In each instance, the
steady state rate of the overall reaction is equal to the velocity of
CPSase. Moreover, the rate constant for the steady state formation of
the final product (carbamoyl aspartate or citrulline) is less than both the reciprocal relation time and the rate constant for the second enzyme in the reaction sequence (ATCase or OTCase). Although the 2-fold
lower transient time at 70 °C compared with 37 °C in this experiment is less than the 5-fold reduction obtained from the progress
curve (Fig. 3 and Table I), channeling, nevertheless, appears to be
more efficient at the higher temperature.
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Table II
Kinetic parameters of the CPSase-ATCase and CPSase-OTCase-coupled
reactions in P. abyssi
The kinetic parameters are: v, the steady-state velocity of
the coupled reaction catalyzed by E1 and
E2; vE1, the steady-state
velocity of the reaction catalyzed by the first enzyme,
E1; 1/ obs, the reciprocal of the apparent
transient time of the coupled reaction; k 2, the
pseudo-first order reaction rate of the second reaction;
k 2(1), the pseudo-first order reaction
rate of the second reaction in the presence of E1.
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Partitioning of Carbamoyl Phosphate between ATCase and
OTCase--
To establish whether endogenously synthesized carbamoyl
phosphate is preferentially utilized by the arginine or pyrimidine biosynthetic pathways, the incorporation of
[14C]bicarbonate into carbamoyl aspartate and citrulline
was measured at 37 °C in cell extracts supplemented with saturating
levels of aspartate, ornithine, and all of the CPSase substrates.
Parallel experiments in which either aspartate or citrulline was
omitted from the assay were simultaneously conducted. Under these
conditions the endogenously synthesized carbamoyl phosphate was
equally (50 ± 5%) partitioned between carbamoyl aspartate and
citrulline, demonstrating that when the enzymes are present in the
proportions found in the cell, carbamoyl phosphate is partitioned
equally between pyrimidine and arginine biosynthetic pathways. The
overall rate of the coupled reactions is limited by the velocity of
CPSase. Consequently, the rate of formation of citrulline or carbamoyl aspartate, upon omission of aspartate or ornithine, respectively, was
comparable to the combined rate when all substrates were present.
Effect of N-(Phosphonacetyl)-L-aspartate--
PALA, a
compound designed as bisubstrate analog of carbamoyl phosphate and
aspartate, effectively inhibits P. abyssi ATCase (24).
Previous kinetic studies (24) showed that PALA is a competitive
inhibitor (Ki = 1.1 µM) with respect
to carbamoyl phosphate. The ATCase activity was reduced by 50% at 300 nM PALA (Fig. 4), when the
assays were conducted in the presence of 5 mM exogenous
carbamoyl phosphate. In contrast, when endogenous carbamoyl phosphate
was used as a substrate by conducting the assays in the presence of
saturating concentrations of NaHCO3, MgATP,
NH4Cl, and aspartate, the concentration of PALA required for 50% inhibition in the rate of formation of carbamoyl aspartate was
reduced to 50 nM.

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Fig. 4.
Influence of PALA on the P. abyssi
ATCase reaction and CPSase-ATCase-coupled reaction. The
effect of increasing concentrations of PALA on the ATCase activity of
P. abyssi cell-free extract (4.5 µg) was determined by
assaying the enzyme using 5 mM [14C]carbamoyl
phosphate and 2 mM aspartate ( ) or using endogenous
synthesized carbamoyl phosphate by initiating the reaction with
[14C]bicarbonate ( ) in the presence of saturating ATP
and NH4Cl and 2 mM aspartate ("Experimental
Procedures").
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Since the binding of PALA and carbamoyl phosphate to the ATCase active
site is competitive, an increase in the apparent affinity for the
inhibitor would be expected for the coupled reaction since the
concentration of carbamoyl phosphate is far below saturation. The
remarkable result is that the magnitude of the effect is so small. The
calculated carbamoyl phosphate concentration of the uncoupled ATCase
reaction in the presence of 50 nM PALA should be 32.6 µM, which is 96-fold higher than that measured in the CPSase-ATCase-coupled reaction (0.34 µM). This result
indicates that the ATCase reaction is much less sensitive to PALA when
carbamoyl phosphate is provided by CPSase than when it is added in the
incubation medium, a result can be explained only by an interaction
between the two enzymes. This interaction would either reduce the
accessibility of the ATCase catalytic site to PALA, increase the
apparent local concentration of carbamoyl phosphate, or it might
increase the affinity of this site for carbamoyl phosphate. This result
strongly suggests that endogenous carbamoyl phosphate is utilized far
more efficiently than the exogenous substrate and provides further evidence for the channeling of the intermediate between the ATCase and
CPSase active sites.
Stable Complex Formation--
Since channeling is presumed to
involve the physical association of the enzymes involved in the
process, size exclusion chromatography was used in an attempt to
demonstrate the formation of a complex between CPSase and either
ATCase, OTCase, or both. The lowest concentration of protein in the
column was 2.5 mg/ml, a value at least 15-fold higher that the
concentration in the assays. At 23 °C in the absence of substrates,
the three enzymes were well separated in the elution profile of a
Sephacryl S300 column (Fig.
5A), suggesting that stable
complexes are not formed.

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Fig. 5.
Size exclusion chromatography of P. abyssi cell-free extracts. The P. abyssi
cell-free extract was subjected to gel filtration on a Sephacryl S300
Superfine column and fractions were assayed for CPSase ( ), ATCase
( ), and OTCase (continuous line) activities as described
under "Experimental Procedures." Chromatography was carried out
under various conditions. Panel A, elution with buffer A at
23 °C; Panel B, elution with buffer A at 37 °C;
Panel C, elution with buffer A at 70 °C; Panel
D, elution with buffer A containing 50 mM carbamoyl
phosphate at 23 °C; Panel E, elution with buffer A
containing all the CPSase and ATCase substrates, at 23 °C;
Panel F, elution with buffer A containing all the CPSase and
ATCase substrates, at 37 °C. The volume of the fractions were 1.0 ml
for the experiments in Panels A, D, and E and
1.70 ml for Panels B, C, and F.
|
|
Since channeling was found to be temperature dependent, the same
experiment was conducted (Fig. 5, B and C) at 37 and 70 °C. Except for small temperature dependent changes in elution
volume, the profile are the same indicating that the enzymes are not
associated and that their molecular mass is unaltered at elevated temperature.
The possibility that substrates or intermediates may promote the
formation of multienzyme complexes was also investigated by
equilibrating and eluting the chromatographic column with various ligands in the elution buffer. Neither 50 mM carbamoyl
phosphate at 23 °C (Fig. 5D), nor saturating
concentrations of all of the CPSase and ATCase substrates at 23 °C
(Fig. 5E) and 37 °C (Fig. 5F), nor the 10 µM
N-(phosphonacetyl)-L-aspartate at 23 °C
promoted stable interactions between the enzymes.
 |
DISCUSSION |
Since the kinetics of P. abyssi OTCase have not been
previously investigated, a preliminary characterization of the
enzyme was a prerequisite for the channeling studies. The
Km for carbamoyl phosphate measured for this enzyme,
8.0 µM, is 45-fold lower than the value obtained of 360 µM obtained (36) for E. coli OTCase. The
[S]0.5 for the carbamoyl phosphate for the
P. abyssi ATCase (24) is also 3-fold lower than its
mesophilic counterpart, perhaps reflecting the necessity for efficient
binding at the elevated temperatures at which these hyperthermophilic enzymes normally function. In this regard, it is interesting that the
apparent affinity of P. abyssi OTCase for ornithine is 8- and 38-fold higher than the values obtained for the E. coli
(37) and yeast (13) enzymes, respectively.
Carbamoyl phosphate is a highly unstable, but essential substrate for
both pyrimidine and arginine biosynthesis. The half-life of carbamoyl
phosphate in an aqueous environment has been determined (6-7) to be
2-3 s at 96 °C. Moreover, it decomposes to cyanate, a promiscuous
carbamylating agent that is highly toxic to most cells. Consequently,
hyperthermophilic organisms would be expected to have evolved
mechanisms to minimize the intracellular concentration of carbamoyl
phosphate or otherwise protect the intermediate at elevated
temperatures. Channeling or molecular compartmentation may represent
one such mechanism for sequestering carbamoyl phosphate and directing
it from its site of synthesis to either ATCase or OTCase where it can
be efficiently and rapidly utilized. There is precedent for partial
channeling of pyrimidine biosynthetic intermediates in
Saccharomyces cerevisiae (11-13), Neurospora
crassa (14-15), and mammals (16, 17, 38). Moreover, there is
convincing evidence for carbamoyl phosphate channeling from CPSase I to
OTCase in rat liver mitochondria (22, 23) in Thermus
aquaticus Z05 (39), a hyperthermophilic eubacteria, and
Pyrococcus furiosus (45).
In this study, four lines of evidence support the contention that
carbamoyl phosphate is channeled in the hyperthermophilic archaeon
P. abyssi. 1) The concentration of exogenous carbamoyl phosphate required to dilute the incorporation of endogenously synthesized intermediate exceeds the [S]0.5 of
ATCase and Km of OTCase by 2- and 37-fold,
respectively; 2) the transient time for the steady state rate of
carbamoyl aspartate and citrulline formation in the
CPSase-ATCase-coupled reaction is significantly lower than would be
observed in the absence of channeling, 3) steady state kinetics of both
coupled systems satisfy the criteria devised by Ovádi et
al. (26) for leaky channeling of carbamoyl phosphate, and 4) PALA
is much less effective inhibitor of the CPSase-ATCase-coupled reaction
than when carbamoyl phosphate is provided in the uncoupled reaction.
The effect of PALA on the rate of carbamoyl aspartate formation on the
CPSase-ATCase-coupled reaction at 37 °C is especially interesting.
Similar anomalous effect on the kinetics of rat liver and P. furiosus OTCase have been observed for the inhibitors PALO (22,
45) and norvaline (23). It could be argued that in the coupled
reaction, access of the inhibitor, but not the substrate aspartate, to
the ATCase active site is restricted due to the formation of a complex
between CPSase and ATCase. However, a more plausible explanation is
that the effective concentration of the intermediate in the vicinity of
the ATCase active site is much higher than the overall concentration of
the intermediate in the system. Assuming that this interpretation is
correct, the effective concentration of carbamoyl phosphate at the
ATCase active site can be calculated from the measured kinetic
parameters. The calculated value is 96-fold higher than the overall
concentration of carbamoyl phosphate in the system, suggesting that the
intermediate is concentrated to an extraordinary extent at the ATCase
active site. The channeling exhibited by the hyperthermophilic enzymes,
even at ambient temperature, is far more efficient than that observed
by the mesophilic enzymes. In the mammalian pyrimidine biosynthetic
complex CAD (17), for example, the effective concentration of carbamoyl
phosphate at the ATCase active site is only 2-fold higher than in the
external phase.
A remarkable discovery was the effect of temperature on the
compartmentation of carbamoyl phosphate. At lower temperatures, channeling is partial or leaky in the sense that some of the carbamoyl phosphate diffuses out of the complex and accumulates in the aqueous milieu. Moreover, while the transient time is much less than that expected for an unlinked system, there was nevertheless a distinct lag
phase prior to reaching the steady state rate of carbamoyl aspartate
formation. The instability of carbamoyl phosphate precluded carrying
out isotope dilution experiments at elevated temperature, but it was
possible to measure the progress curves for carbamoyl aspartate
formation. While the 2-5-fold decrease in transient time at 70 °C
suggests an improvement of channeling efficiency compared with
37 °C, the most striking results were obtained at 90 °C. At this
temperature, both the transient time and the steady state concentration
of carbamoyl phosphate were undetectable, indicating that channeling
becomes absolute at temperatures close to the growth optimum of
P. abyssi.
P. abyssi has a single CPSase which provides carbamoyl
phosphate for both pyrimidine and arginine biosynthesis and thus, the way in which the intermediate is partitioned between the two competing pathways is of interest. The intracellular concentrations of the enzymes in exponentially growing P. abyssi cells is such
that in the presence of saturating substrates, the rate of formation of
carbamoyl aspartate and citrulline are nearly the same. Carbamoyl phosphate channeling occurs in both pathways with a comparable efficiency, although the process has so far only been studied for the
CPSase-OTCase-coupled reaction at 37 °C. Taken together, these
observations suggest that the flux of metabolites through the
pyrimidine and arginine biosynthetic pathways is potentially equivalent. The partitioning experiments reported here, which show that
endogenously synthesized carbamoyl phosphate is distributed equally to
ATCase and OTCase, support this interpretation. The equivalence of the
pathways, despite a 7.5-fold lower carbamoyl phosphate
Km of OTCase as compared with ATCase, suggests that
the flux is determined by channeling rather than the absolute affinity
of the second enzyme for the intermediate. In the cell, the relative
activity of the pyrimidine and arginine pathways must be regulated by
the availability of the second substrate, aspartate and citrulline, and
probably to a greater extent by the concentration of allosteric effectors.
At the molecular level, channeling usually involves the direct
interaction between the enzymes that participate in the process. The
most often cited paradigm is the bifunctional complex tryptophan synthetase (8, 9) which channels indole through a 25-Å long tunnel
that bridges its site of synthesis on the
subunit to the
subunit where tryptophan is synthesized. Recent structural studies (40)
of glutamine PRPP amidotransferase have shown that intermediate ammonia
is sequestered within a hydrophobic cavity formed at the interface of
the amidotransferase and synthetase subunit. Most remarkably (41), the
intermediates involved in carbamoyl phosphate synthesis are believed to
traverse a distance of 96 Å through a long tunnel which spans the
interior of E. coli CPSase. In each of these examples a
stable physical complex is formed, although there are many other
examples (42, 43) for which there is compelling kinetic evidence for
channeling in dynamic enzyme complexes.
Given the observation that a single CPSase must service two distinct
biosynthetic pathways in P. abyssi, there are three possible scenarios that could account for channeling: 1) CPSase forms two different stable complexes with ATCase and OTCase, 2) CPSase forms a
tertiary complex with both ATCase and OTCase, and 3) complex formation
is dynamic in the sense that CPSase is transiently associated with each
of these acceptors and that intermediate transfer occurs during short
lived contact. The results reported here failed to detect a stable
complex between any of these enzymes suggesting that complex formation
is either weak or transient. Neither elevated temperature, which
promotes more efficient channeling, nor the presence of substrates
which could be envisioned to alter the enzyme conformation and thus,
promote complex formation, lead to the formation of complexes which
could be isolated by gel filtration. Dynamic channeling requires (44) a
high concentration of the binary enzyme complex. However, a rapid rate
of dissociation would be expected to allow dissociation of the complex
on the column. Alternative more sensitive approaches may be required to
detect the interactions that are likely to occur.
In summary, the channeling of carbamoyl phosphate in P. abyssi sequesters the intermediate, reduces its steady state
concentration in the cell, and provides for its efficient utilization
by both CPSase and OTCase and, thus, may be an important mechanism for preservation of this thermolabile intermediate at the extreme temperatures at which this hyperthermophilic organism flourishes.