Channeling of Carbamoyl Phosphate to the Pyrimidine and Arginine Biosynthetic Pathways in the Deep Sea Hyperthermophilic Archaeon Pyrococcus abyssi*

Cristina PurcareaDagger §, David R. EvansDagger , and Guy Hervé§

From the Dagger  Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201 and the § Laboratoire de Biochimie des Signaux Régulateurs Cellulaires et Moléculaires, UMR CNRS 7631, Université Pierre et Marie Curie, 96, Bd. Raspail, 75006 Paris, France

    ABSTRACT
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Abstract
Introduction
References

The kinetics of the coupled reactions between carbamoyl-phosphate synthetase (CPSase) and both aspartate transcarbamoylase (ATCase) and ornithine transcarbamoylase (OTCase) from the deep sea hyperthermophilic archaeon Pyrococcus abyssi demonstrate the existence of carbamoyl phosphate channeling in both the pyrimidine and arginine biosynthetic pathways. Isotopic dilution experiments and coupled reaction kinetics analyzed within the context of the formalism proposed by Ovádi et al. (Ovádi, J., Tompa, P., Vertessy, B., Orosz, F., Keleti, T., and Welch, G. R. (1989) Biochem. J. 257, 187-190) are consistent with a partial channeling of the intermediate at 37 °C, but channeling efficiency increases dramatically at elevated temperatures. There is no preferential partitioning of carbamoyl phosphate between the arginine and pyrimidine biosynthetic pathways. Gel filtration chromatography at high and low temperature and in the presence and absence of substrates did not reveal stable complexes between P. abyssi CPSase and either ATCase or OTCase. Thus, channeling must occur during the dynamic association of coupled enzymes pairs. The interaction of CPSase-ATCase was further demonstrated by the unexpectedly weak inhibition of the coupled reaction by the bisubstrate analog, N-(phosphonacetyl)-L-aspartate (PALA). The anomalous effect of PALA suggests that, in the coupled reaction, the effective concentration of carbamoyl phosphate in the vicinity of the ATCase active site is 96-fold higher than the concentration in the bulk phase. Channeling probably plays an essential role in protecting this very unstable intermediate of metabolic pathways performing at extreme temperatures.

    INTRODUCTION
Top
Abstract
Introduction
References

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.

    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 Delta pro-lac Delta 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 (tau ) 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,
[I]<UP>normalized</UP>=[I]<UP>observed</UP><FR><NU>(V<SUB>o</SUB> <UP>extract, 37 °C</UP>)</NU><DE>(V<SUB>o</SUB> <UP>observed</UP>)</DE></FR> (Eq. 1)
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.
S <LIM><OP><ARROW>→</ARROW></OP><LL>E<SUB>1</SUB></LL><UL>k<SUB>1</SUB></UL></LIM> <UP>I</UP> <LIM><OP><ARROW>→</ARROW></OP><LL>E<SUB>2</SUB></LL><UL>k<SUB>2</SUB></UL></LIM> P (Eq. 2)

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,
V<SUB>E1</SUB>=V<SUB>o</SUB> <UP>and</UP> 1/&tgr;<SUB><UP>obs</UP></SUB>>k<SUB>&Ggr;2</SUB><SUP>(1)</SUP>≤k<SUB>&Ggr;2</SUB> (Eq. 3)
where k&Ggr;2(1) and k&Ggr;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 (triangle ), 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 (open circle ). 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.

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).

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 (tau ), 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 tau  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 tau  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 tau  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.

                              
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Table I
Transient time parameters

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 (open circle ), 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.

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/tau obs), the pseudo first-order rate constant of the individual reaction catalyzed by E2 alone (k&Ggr;2) and in the presence of E1 (k&Ggr;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/tau obs, the reciprocal of the apparent transient time of the coupled reaction; k&Ggr;2, the pseudo-first order reaction rate of the second reaction; k&Ggr;2(1), the pseudo-first order reaction rate of the second reaction in the presence of E1.

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 (open circle ) 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").

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 (open circle ), 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 alpha  subunit to the beta  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.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM-74399 (to D. E.), by the Center National de la Recherche Scientifique, Université Pierre et Marie Curie, Paris, and a Fellowship from the French Government (to C. P.).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.

2 During the course of the 30-min incubation approximately 40% of the exogenous carbamoyl phosphate is hydrolyzed. Thus, the mean concentration of carbamoyl phosphate is 20% lower than the nominal concentration on the x axis. Taking this factor in consideration, the dilution profiles of both the coupled system and uncoupled control would be shifted by 20% to lower concentrations but the shape of the curves would not be significantly altered.

3 It can be calculated that the transient time represents 0.67 of the time required for the system to reach steady state.

    ABBREVIATIONS

The abbreviations used are: CPSase, carbamoyl-phosphate synthetase; ATCase, aspartate transcarbamoylase; OTCase, ornithine transcarbamoylase; PALA, N-(phosphonacetyl)-L-aspartate.

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Top
Abstract
Introduction
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