©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Substructure of the Amidotransferase Domain of Mammalian Carbamyl Phosphate Synthetase (*)

(Received for publication, September 8, 1994; and in revised form, November 23, 1994)

Hedeel I. Guy David R. Evans

From the Department of Biochemistry, Wayne State University School of Medicine, Detroit, Michigan 48201

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The amidotransferase or glutaminase (GLNase) domain of mammalian carbamyl phosphate synthetase (CPSase), part of the 243-kDa CAD polypeptide, consists of a carboxyl half that is homologous to all trpG-type amidotransferases and an amino half unique to the carbamyl phosphate synthetases. The two halves of the mammalian GLNase domain have been cloned separately, expressed in Escherichia coli, and purified. The 21-kDa carboxyl half, the catalytic subdomain, is extraordinarily active. The k is 347-fold higher and the K is 40-fold lower than the complete GLNase domain. Unlike the GLNase domain, the catalytic subdomain does not form a stable hybrid complex with the E. coli CPSase synthetase subunit. Nevertheless, titration of the synthetase subunit with the catalytic subdomain partially restores glutamine-dependent CPSase activity. The 19-kDa amino half, the interaction subdomain, binds tightly to the E. coli CPSase large subunit. Thus, the GLNase domain consists of two subdomains which can autonomously fold and function. The catalytic subdomain weakly interacts with the synthetase domain and has all of the residues necessary for catalysis. The interaction subdomain is required for complex formation and also attenuates the intrinsically high activity of the catalytic subdomain and, thus, may be a key element of the interdomain functional linkage.


INTRODUCTION

Amidotransferases catalyze glutamine hydrolysis to supply ammonia for de novo biosynthesis of purines, pyrimidines, glucosamine, folate, nicotinamide, and several amino acids(1, 2) . There are two classes of amidotransferase, trpG and purF types(2) , which have dissimilar amino acid sequences but a common catalytic mechanism(3) . In both types of enzymes, the amidotransferase activity is usually associated with a separate subunit or domain of the biosynthetic complex. The trpG amidotransferase domains represent a homologous family of proteins with a molecular mass of 20 kDa and a sequence that includes three nearly invariant regions (regions I-III) shown to be important for the function of the molecule(2, 3) .

Carbamyl phosphate synthetase (CPSase, (^1)EC 6.3.5.5), a trpG type amidotransferase, (^2)catalyzes the formation of carbamyl phosphate from bicarbonate, 2 mol of ATP, and glutamine(4) . Escherichia coli carbamyl phosphate synthetase (5) consists of a small (42-kDa) subunit which carries glutaminase activity, and a large (118-kDa) subunit which catalyzes ammonia-dependent carbamyl phosphate synthesis. The structural organization of the enzyme is quite different in mammals. De novo pyrimidine biosynthesis is initiated by CAD(6, 7, 8) , a large multifunctional protein which catalyzes the first three steps of the pathway. The 243-kDa CAD polypeptide is organized into separate structural domains (9, 10, 11) which have carbamyl phosphate synthetase activity, aspartate transcarbamylase (EC 2.1.3.2), and dihydroorotase activities (EC 3.5.2.3). Controlled proteolysis studies(9, 10, 11, 12, 13, 14) and determination of the amino acid sequence of the hamster CAD polypeptide (15, 16, 17, 18) showed that carbamyl phosphate synthesis involves two distinct domains, a 120-kDa CPSase synthetase domain and a 40-kDa glutaminase (GLNase) domain. Both of the mammalian domains have a strong sequence similarity (40-50%) to their counterparts in E. coli and other species(17) .

The E. coli CPSase subunits have been dissociated and shown to function autonomously(5) . However, there is extensive evidence(5, 19, 20, 21, 22, 23, 24) for a functional linkage between the glutamine binding site of the small subunit and the active site of the large synthetase subunit which modulates their activities. Glutamine hydrolysis does not proceed at a significant rate in the absence of bicarbonate and ATP. This functional linkage, which minimizes the hydrolysis of glutamine when the other substrates needed for carbamyl phosphate synthesis are limiting, is also present in CAD. ATP and bicarbonate increase the k of the glutaminase reaction 14-fold by increasing the rate of hydrolysis of the thioester intermediate (25) formed during the catalytic cycle.

We have recently cloned the glutaminase domain of CAD and expressed the protein in E. coli(26) . The purified protein had barely detectable catalytic activity but formed a fully functional hybrid with the E. coli CPSase synthetase domain indicating that the functional linkage between the glutaminase and synthetase domains had been restored. This observation suggests that the subunit interfaces must be nearly identical in the eukaryotic and prokaryotic proteins and that the interdomain linkage has been highly conserved throughout the course of evolution.

The glutaminase domain of all carbamyl phosphate synthetases is unusual in that it consists of a 21-kDa carboxyl half which is homologous to all of the trpG-type amidotransferases and a 19-kDa amino half which occurs only in this family of molecules(17, 27, 28, 29, 30, 31, 32, 33) . The carboxyl half or catalytic subdomain contains the active site cysteine that participates in glutamine hydrolysis. The function of the amino half of the CPSase glutaminase domain is unknown, although, in a recent study (34) , a series of point mutations and deletions were introduced into the E. coli CPSase small subunit. Removal of the amino-terminal third (residues 1-113) of the glutaminase subunit prevented the association with the CPSase synthetase subunit, suggesting that this region is required to stabilize the complex. Thus, the amino half of the glutaminase domain has been designated the interaction subdomain.

As reported here, we have cloned and expressed the two halves of the hamster CAD glutaminase domain to determine whether they are separately folded subdomains with distinct functions. This study showed that all of the residues required for glutamine binding and hydrolysis are present in the catalytic domain and that the interaction domain, in addition to its role in maintaining the physical association of the glutaminase and synthetase domain, also participates in the interdomain signaling.


EXPERIMENTAL PROCEDURES

Plasmids and Strains

The 7.1-kb plasmid, pHG-GLN52(26) , consists of a 1.1-kb insert encoding the CAD glutaminase domain under the control of the pyrBI promoter(36) . The 4.9-kb plasmid, pJLA503(38) , contains the P(R), P(L) promoters and the CI gene. The E. coli host strains used were EK1104(36) , which lacks E. coli aspartate transcarbamylase, and L673 which is defective in carAB and the Lon protease(37) .

Cell Growth and Recombinant DNA Methods

For induction of the recombinant proteins under control of the pyrBI promoter, the transformants were grown, induced, and harvested as described previously(26) . For induction of recombinant proteins under control of the promoters and the CI gene, the transformed EK1104 or L673 cells were grown in 2 times YT media supplemented with 5 mM K(2)PO(4), pH 7.0, 28 mM glucose, and 100-150 mg/liter ampicillin. The media were innoculated with an overnight culture (1:50, v/v) and grown at 28 °C until the cells reached early log phase (A of 0.3-0.4). An equal volume of media preheated to 65 °C was then added, and the cells were grown at 42 °C for 2-3 h, then harvested by centrifugation at 2,700 times g for 30 min at 4 °C. Plasmid isolation(39) , transformations(40) , purification of DNA fragments, restriction digests, ligations, and other DNA methods (39) were the same as those used (26) for construction of pHG-GLN52.

Protein Methods

Native CAD was isolated from an overproducing strain of Syrian hamster cells) as described previously(8, 41) . The E. coli CPSase large subunit was isolated from the high copy number plasmid, pHN12(26, 42) . Protein determination(43) , SDS-polyacrylamide gel electrophoresis(44) , immunoblotting(12, 45, 46) , and sucrose gradient centrifugation were carried out as described previously(26) .

Enzyme Assays

Carbamyl phosphate synthetase activity was assayed by a radiometric procedure(8, 9, 26) . Glutaminase activity was measured by a thin layer chromatography method(47) , by a coupled spectrophotometric assay(48) , or by HPLC separation of the reactant glutamine from the product glutamate as described previously (25) except that the column was eluted with 70% 50 mM sodium acetate, pH 5.9, 30% methanol. The program Minsq (Micromath) was used to obtain a nonlinear least squares fit of the saturation curves to the Michaelis-Menten equation.

Purification of the CAD GLN Catalytic Subdomain

A 500-ml culture of pHG-GLN15-transformed L673 cells yielded 5.3 g of cells. The cells were suspended in 30 ml of 0.05 M Tris-Cl, pH 7, 1 mM DTT, 0.2 mM phenylmethylsulfonyl fluoride, 5% glycerol (sonication buffer) and sonicated on ice twice for 2 min as described previously(26) . The sonicate was centrifuged at 29,000 times g for 30 min at 4 °C, and the supernatant (30 ml) was applied to a 50-ml DEAE-Sephacryl column equilibrated in sonication buffer and eluted with a linear gradient of 0-1 M NaCl in the same buffer. Fractions containing the GLN catalytic subdomain, identified by SDS-gel electrophoresis and immunoblotting, were pooled, precipitated with 55% ammonium sulfate, and fractionated on a Sephadex G-200 column and eluted with 0.1 M potassium phosphate, pH 7.6, 1 mM DTT, 1 mM EDTA, and 5% glycerol. Fractions containing the homogeneous protein were pooled and stored at -0 °C.

Purification of the CAD GLN Interaction Subdomain

A 500-ml culture of pJHN-GLN17-transformed L673 cells yielded 3.1 g of cells. The cells were suspended in 20 ml of sonication buffer and sonicated as described above. The sonicate was centrifuged, and the supernatant (20 ml) was applied to a 50-ml DEAE-Sephacryl column and eluted with a 0-0.3 M NaCl gradient in sonication buffer. The nearly homogeneous interaction subdomain eluted at approximately 0.10 M NaCl. The pooled fractions were stored at -70 °C.


RESULTS

Construction of the Recombinant CAD GLN Subdomains

The glutaminase domain, located at the amino end of the CAD polypeptide (Fig. 1), consists of two halves defined (17) by homology with other amidotransferases. The amino half, designated the interaction domain, extends from residues 1 to 174, while the carboxyl half or catalytic domain begins at residue 175 and extends to residue 365, the last amino acid before the start of the linker which connects the GLNase and CPSase domains.


Figure 1: Subdomain structure of the CAD glutaminase domain. The glutaminase domain consists of residues 1-366 on the amino end of the CAD polypeptide. The 40-kDa domain is composed of two putative subdomains, the interaction and the catalytic subdomains. The interaction domain extends from residue 1 to 174. The catalytic subdomain, residues 175 to 365, contains three highly conserved regions (I, II, and III) that are common to all amidotransferases. Region IV is conserved only in the CPSases, but not found in other amidotransferases(2, 3) . Controlled proteolysis (14) cleaves the catalytic subdomain at residue 287 (protease site 1) more rapidly than the linker (protease site 2) connecting the GLNase and CPSase domains, suggesting that the catalytic subdomain may itself be composed of two smaller subdomains.



The recombinant, pJHN-GLN17, encoding the amino half of the hamster CAD glutaminase domain (Fig. 2), was obtained by deletion of pHG-GLN52(26) , a plasmid which codes for the entire 40-kDa glutaminase domain. The parent plasmid was restricted with StyI and EcoRI, and the 5` extensions were made flush using the Klenow fragment. The 3.9-kb fragment was gel-purified and religated to yield pJHN-GLN17, which contained a 539-bp cDNA insert encoding residues 1 to 180 of the CAD polypeptide. The coding sequence, thus, extends 6 residues into the putative catalytic subdomain. In addition, 3 residues derived from the vector are added to the carboxyl end of the protein. Expression of the recombinant, like the parental plasmid, is under control of the pyrBI promoter.


Figure 2: Cloning of the CAD GLNase interaction and catalytic subdomains. To clone the interaction subdomain (on the left), pHG-GLN52(26) , a plasmid encoding the CAD GLNase domain (dark-shaded area) under the control of the pyrBI promoter (reg), was restricted with StyI and EcoRI, and the 5` extensions were made flush using the Klenow fragment of DNA polymerase. The resulting 3.9-kb plasmid pJHN-GLN17 contained a 539-bp cDNA fragment encoding the entire interaction subdomain and six amino acid residues of the amino end of the catalytic subdomain. In addition, three amino acids (-Leu-Asn-Ser-COOH) derived from the vector are encoded prior to the stop codon. The recombinant, pJHN-GLN17, is under control of the pyrBI promoter. To clone the catalytic subdomain (on the right), a 565-bp XhoII-SmaI fragment (dark-shaded area) was excised from the recombinant plasmid pCAD142 (35) which contains most of the CAD coding sequence. The fragment was inserted into the BamHI and EcoRI sites of the vector pJLA503 after restriction and removal of the 5` extensions of EcoRI cleavage site with mung bean nuclease. The resulting recombinant pHG-GLN15 is under the control of the tandem phage promoters (P(R), P(L)) and has a temperature-sensitive repressor gene (CI) which allows expression by heat shock. The plasmid encodes residues 175 to 365 of CAD and has five amino acids (NH(2)-Met-His-Arg-Asn-Arg-) and six amino acids (-Arg-Arg-Pro-Arg-Cys-Pro-COOH) derived from the vector appended to the amino and carboxyl ends of the protein, respectively.



The sequence encoding the carboxyl half of the GLNase domain was excised from the partial cDNA clone pCAD142(35) , by restriction with XhoII and SmaI (Fig. 2). The insert was cloned into the plasmid, pJLA503, a heat shock vector under control of dual promoters. The vector was cut with EcoRI and then with BamHI after removal of the 5`-EcoRI extensions with mung bean nuclease. The resulting recombinant, pHG-GLN15, has a 565-bp insert corresponding to residues 175 to 365 of the CAD polypeptide. When expressed, the recombinant protein has 5 and 6 amino acid residues derived from the vector added to the amino and carboxyl ends, respectively.

Expression and Purification of the CAD GLN Subdomains

The uracil-dependent E. coli carAB-deficient strain, L673, which lacks both the glutaminase and synthetase subunits of E. coli CPSase, served as the host for expression of the mammalian GLNase subdomains.

For expression of the interaction subdomain, pJHGLN-17 transformants were grown in minimal media containing limiting uracil (12 µg/ml). The pyrBI promoter is derepressed when the uracil is exhausted (16-18 h), and a new 19-kDa protein began to accumulate in the extracts. This species was not found in extracts of cells transformed with the vector, pEK81. Immunoblots (Fig. 3) probed with hamster CAD antibodies confirmed that this polypeptide was of mammalian origin. The interaction subdomain was purified by DEAE-Sephacryl chromatography. We typically obtained 1-2 mg of the nearly homogeneous protein from a 500-ml culture of transformed cells.


Figure 3: Expression and purification of the CAD GLNase interaction subdomain. The plasmid pJHN-GLN17, which encodes the GLNase interaction subdomain, was transformed into the strain EK1104 (lane 1) and the Lon protease-deficient strain L673 (lane 2). The induction followed the protocol described under ``Experimental Procedures.'' Cell extracts were prepared by suspending 6 ml of packed cells (50 mg of cell protein) in 0.1 ml of 5 times SDS-sample buffer and 0.4 ml of 50 mM Tris HCl, pH 7.4, 5% glycerol, 1 mM DTT. The suspension was immediately heated to 85 °C for 15 min, then sonicated briefly, and 50-µl aliquots were loaded onto a 7.5-15% gradient SDS-gel. This figure shows the SDS-polyacrylamide gel (panel A) and the corresponding immunoblot (panel B) of the pJHN-GLN17/EK1104 (lane 1), pJHN-GLN17/L673 (lane 2) transformants, and EK1104 and L673 cells transformed with the vector only (lanes 3 and 4, respectively). An SDS-polyacrylamide gel of 1 µg of partially purified interaction domain (panel C) is also shown.



For the expression of the catalytic subdomain, pHG-GLN15-transformed cells were induced by heat shock (see ``Experimental Procedures''). SDS-gel electrophoresis showed that extracts of pHG-GLN15 transformants contained a new species with a molecular mass of 21 kDa, that was not present in extracts of cells transformed with the vector. The recombinant protein reacted specifically with CAD antibodies indicating that it was part of the CAD polypeptide. Moreover, extracts of cells transformed with pHG-GLN15, but not the untransformed cells, had high glutaminase activity (Table 1). The protein was purified 667-fold (Table 1) by chromatography (Fig. 4) on a DEAE-Sephacryl column followed by Sephadex G-200 gel filtration. A 500-ml culture usually yielded 30 µg of nearly homogeneous catalytic subdomain.




Figure 4: Expression and purification of the CAD GLNase catalytic subdomain. The recombinant, pHG-GLN15, which encodes the GLNase catalytic subdomain, was transformed into the Lon protease-deficient E. coli strain, L673(37) , and induced as described under ``Experimental Procedures.'' Cell extracts were prepared as described in the legend to Fig. 3, from 6 ml of packed cells (60 mg of cell protein). The expression of the 21-kDa catalytic subdomain at 35, 90, and 150 min after heat shock (lanes 1-3) was shown by SDS-gel electrophoresis (panel B) and immunoblotting (panel D) of the pHG-GLN15 transformants. This species was not seen in extracts of cells transformed with the vector (lanes 4-6). For comparison, the SDS-polyacrylamide gel (panel A) and immunoblot (panel C) of EK1104 cells transformed with pHG-GLN52, which encodes the 40-kDa full-length GLNase domain, are also shown as is an SDS gel of 0.2 µg of the purified GLNase catalytic subdomain (panel E).



Glutaminase Activity of the Purified GLNase Catalytic Subdomain

The isolated full-length GLNase domain was found to have barely detectable glutaminase activity(26) . Using the more sensitive HPLC assay method, a glutamine saturation curve was obtained which gave the kinetic parameters summarized in Table 2. The K(m) for glutamine was 46-fold higher, and the k was 8.5-fold lower than the corresponding values of CAD measured in the absence of ATP and bicarbonate.



In contrast, the purified catalytic subdomain was hyperactive (Fig. 5). Excision of the interaction domain increased the k to 5.73 s, a value that is 347-fold higher than that of the isolated full-length GLNase domain, and restored the K(m) to the normal value observed for the intact CAD complex. As expected, the isolated GLNase interaction domain had no detectable catalytic activity.


Figure 5: Glutamine saturation curve of the GLNase catalytic subdomain and of the GLNase activity of CAD. The activity (bullet-bullet) of the purified GLNase catalytic subdomain (0.2 µg) was assayed by the spectrophotometric method (see ``Experimental Procedures'') as a function of glutamine concentration. For comparison, the glutamine saturation curve of 30 µg of CAD (circle-circle) is shown. In addition, a saturation curve given by 30 µg of the isolated recombinant GLNase domain was also obtained using the more sensitive HPLC method but is not shown here. The kinetic parameters obtained for all three proteins are summarized in Table 2.



Gel Filtration of the GLNase Catalytic Subdomain in the Presence of the E. coli CPSase Synthetase Subunit

Our previous studies (26) showed that, remarkably, the isolated CAD GLNase domain forms a fully functional, 1:1 stoichiometric complex with the E. coli synthetase large subunit. The complex is very stable and could be isolated by gel filtration chromatography. To determine whether the catalytic subdomain can also form a hybrid complex, the purified subdomain was mixed at a molar ratio of approximately 1:1 with the purified E. coli CPSase synthetase subunit and applied to a Sephacryl S-300 column. SDS-polyacrylamide gel electrophoresis and assays of the NH(3)-dependent CPSase activity showed (Fig. 6) that the 120-kDa E. coli CPSase subunit eluted appreciably ahead of the 21-kDa catalytic subdomain indicating that a complex was not formed.


Figure 6: Gel filtration of the GLNase catalytic subdomain and the E. coli CPSase synthetase subunit. The purified E. coli synthetase large subunit (25 µg) was incubated with the purified catalytic subdomain (5 µg) in 0.1 M KH(2)PO(4)/KOH, pH 7.6, 1 mM EDTA, 1 mM DTT, 5% glycerol for 5 min at 4 °C and then chromatographed on a 2.9 times 93 cm Sephacryl S-300 gel filtration column. The column was pre-equilibrated and eluted with the same buffer. NH(3)-dependent CPSase assays and SDS-gel electrophoresis showed that the E. coli CPSase synthetase subunit eluted in fractions 62 through 76. The SDS gel showed that the catalytic subdomain eluted in fractions 78 through 80.



Titration of E. coli CPSase Synthetase Subunit with the CAD GLN Catalytic Subdomain

Although the GLNase catalytic domain did not form a stable complex with the E. coli CPSase synthetase subunit, functional interactions between these two species were demonstrated (Fig. 7) by titrating the E. coli CPSase synthetase subunit with the purified catalytic subdomain. The isolated E. coli synthetase subunit could not catalyze the formation of carbamyl phosphate from glutamine. However, glutamine-dependent CPSase activity was restored as increasing amounts of the mammalian GLNase catalytic subdomain were added. The curve reached saturation at a molar ratio of GLNase catalytic subdomain to CPSase synthetase subunit of 12. The maximum activity, expressed relative to the amount of synthetase subunit, was 0.191 µmol/min/mg. In contrast, the complete GLNase domain formed a 1:1 stoichiometric complex with the E. coli CPSase synthetase subunit. The hybrid complex was fully active with a specific activity of 3.21 µmol/min/mg at a molar ratio of GLNase/CPSase synthetase of 0.9(26) .


Figure 7: Titration of the E. coli CPSase synthetase subunit. Increasing amounts (0.05-6 µg) of the purified mammalian glutaminase catalytic subdomain were incubated with 1.95 µg of the purified E. coli CPSase synthetase subunit in 0.1 ml of 0.05 M Tris-HCl, pH 7.4, 1 mM DTT, 5% glycerol for 5 min at room temperature. The samples were assayed for glutamine-dependent carbamyl phosphate synthetase activity (bullet-bullet) as described under ``Experimental Procedures.'' The ordinate represents fraction of maximum observed activity. The molar ratio of the catalytic subdomain to the E. coli synthetase subunit was calculated assuming a molecular mass of 20 and 120 kDa, respectively. The titration curve of the E. coli synthetase subunit with the full-length GLNase domain, carried out under identical conditions (circle-circle) taken from Guy and Evans(26) , is also shown for comparison.



This experiment indicated that the interactions between the GLNase catalytic subdomain and the CPSase synthetase domain are relatively weak, and that the complex, once formed, is not fully functional. Glutamine saturation curves of this hybrid (Fig. 8) gave the same K(m) for glutamine, but the k was 17-fold lower than the value obtained for the hybrid formed from the complete GLNase domain.


Figure 8: Kinetics of mammalian E. coli hybrids CPSase. The mammalian catalytic domain (2 µg) was incubated at an equimolar ratio with the E. coli CPSase synthetase subunit (10 µg), and the glutamine-dependent CPSase activity was measured as a function of glutamine concentration (lower panel) at 5 mM [^14C]sodium bicarbonate (0.8 µCi/µmol) and 20 mM ATP. The saturation curve for the hybrid of the full-length mammalian GLNase domain and the E. coli CPSase large subunit (upper panel), taken from Guy and Evans(26) , is shown for comparison. The ordinate represents the specific activity of the complex expressed per mg of the E. coli CPSase synthetase subunit. The V(max) of the hybrid of the mammalian GLNase catalytic subdomain with the E. coli CPSase synthetase subunit was 0.191 µmol/min/mg, while the corresponding hybrid formed with the full-length mammalian GLNase domain is 3.21 µmol/min/mg.



Demonstration of a Mammalian GLNase Interaction Subdomain E. coli CPSase Hybrid

The formation of the mammalian bacterial hybrid was demonstrated by sucrose gradient centrifugation (Fig. 9). When extracts of pJHN-GLN17/L673 transformants were mixed with the purified E. coli CPSase large subunit, a species formed which sedimented more rapidly than the CPSase subunit. The isolated E. coli CPSase subunit was found in fractions 19-22, whereas in the presence of the mammalian domain, it appeared in fractions 15-18. Immunoblotting using CAD antibodies as a probe confirmed that the 19-kDa GLNase interaction subdomain co-sedimented with the E. coli CPSase subunit.


Figure 9: Sucrose gradient of the interaction domain complex with the E. coli CPSase synthetase subunit. To obtain the GLNase interaction subdomain, pJHN-GLN17/L673 transformants were grown as described under ``Experimental Procedures.'' The cell pellet (0.1 g) was resuspended in 0.5 ml of 0.2 M potassium phosphate, pH 7.6, 1 mM EDTA, and sonicated. The extract was then centrifuged at 16,000 times g for 20 min at 4 °C. The supernatant (0.150 ml, 3 mg of protein) was incubated with the purified E. coli synthetase subunit (20 µg) in a total volume of 0.2 ml for 5 min at 25 °C. The reaction mixture was then fractionated by sucrose gradient centrifugation at 190,000 times g for 9 h at 4 °C. The E. coli CPSase synthetase subunit (20 µg) was centrifuged in a parallel gradient. The gradients containing the mixture of the mammalian GLNase interaction subdomain and the E. coli CPSase synthetase subunit (bullet-bullet) and the E. coli CPSase synthetase subunit alone (circle-circle), were then fractionated and assayed for ammonia-dependent CPSase activity as described under ``Experimental Procedures.'' An immunoblot of the hybrid fractions using anti-CAD serum showed the presence of a 19-kDa fragment corresponding to the interaction subdomain co-sedimenting with the E. coli synthetase subunit.




DISCUSSION

When the cDNA sequences encoding the two halves of the CAD amidotransferase domain were cloned and expressed in E. coli, stable, soluble proteins were produced, which, when isolated, were found to have specific functions. This result showed that the glutaminase domain of the CAD CPSase, and, by inference, the other members of this family, consists of two independent subdomains which fold and function autonomously. The chain segment connecting the catalytic and interaction subdomains is probably buried within the molecule since no proteolytic cleavage site was found (14) at the junction between the two subdomains.

The complete CAD GLNase domain has very low glutaminase activity as a consequence of a 46-fold increase in the K(m) and an 8.5-fold lower k compared to the same domain present in the intact CAD complex. Thus, when isolated from the remainder of the molecule, the apparent second order rate constant k/K(m) is reduced 392-fold. Similarly, when the subunits of E. coli CPSase were dissociated(5) , there was a 300-fold increase in the K(m) for glutamine. These authors suggested that there may be additional residues on the large synthetase subunit which are involved in glutamine binding. However, we show here that the isolated catalytic subdomain has the same K(m) for glutamine as the intact CAD complex and a k which exceeds that of CAD activated by ATP and bicarbonate. The differences are even larger when the activity of the catalytic domain is compared to the isolated complete GLNase domain. The k of the catalytic subdomain is 347-fold higher, and the K(m) is 40-fold lower than the corresponding values for the GLNase domain. Thus, excision of the interaction subdomain from the CAD glutaminase results in a 14,000-fold increase in the apparent second order rate constant for glutamine hydrolysis. This result demonstrates that all of the residues necessary for glutamine binding and catalysis are located within the catalytic subdomain. It is more likely that the suppression of the glutaminase activity is a consequence of the functional linkage between the GLNase and CPSase domains that is necessary to avoid the hydrolysis of glutamine when the other substrates required for the synthesis of carbamyl phosphate are limiting.

The interaction domain binds to the E. coli synthetase domain forming a complex which is stable enough to detect by sucrose gradient centrifugation. This result is consistent with the results of Guillou et al.(34) who showed that deletion of 113 residues from the amino end of the E. coli GLNase subunit prevents its association with the synthetase subunit. Thus, the interaction domain is crucial for the formation of a stable complex between the glutaminase and synthetase domains or subunits. In contrast, we were unable to detect a stable complex of the catalytic subdomain and the E. coli CPSase synthetase subunit, indicating that the interaction between these domains are weak. Nevertheless, the titration curve (Fig. 7) and the steady state kinetics (Fig. 8) clearly show that interactions between the catalytic subdomain and the the CPSase synthetase domain do exist.

Analysis of the titration curve suggests that when the mammalian GLNase catalytic subdomain is mixed in a one to one molar ratio with the E. coli CPSase synthetase subunit, only 5% of the E. coli subunits have bound the GLNase domain. Glutamine-dependent CPSase assays measure only the activity of the complex since the free unbound CPSase synthetase has no glutamine-dependent activity. Taking this factor into account, we can calculate the steady state kinetic parameters (Table 3) of the complex between the catalytic subdomain and the synthetase subunit. The K(m) for glutamine is similar to that of the hybrid formed with the complete mammalian GLNase domain. However, the k for the glutamine-dependent CPSase activity is 8.5-fold lower indicating that while the catalytic subdomain binds, albeit tenuously, to the CPSase domain and can transfer ammonia to the synthetase subunit, the functional linkage is not restored.



This study, therefore, shows that the interaction domain has two functions, 1) it forms strong noncovalent interactions with the CPSase synthetase domain that are necessary for the functional interactions, and 2) it interacts with the GLNase catalytic subdomain to decrease glutamine binding and suppress the high activity of the subdomain.

The low affinity for glutamine and a low turnover number of the isolated GLNase domain can thus be attributed to inhibitory effects exerted by the interaction domain. When the glutaminase domain associates with the synthetase domain, this inhibition is partially relieved. The K(m) is restored to normal levels, and the k increases from 0.0165 to 0.140. However, only in the presence of ATP and bicarbonate does the turnover number approach the value observed for the isolated catalytic subunit.

We propose that the interaction domain is an important element of the functional linkage between the CPSase glutaminase and synthetase domains or subunits. In this model, illustrated schematically in Fig. 10, the GLNase domain can exist in two states. In the absence of the other CPSase substrates, ATP and bicarbonate, the domain is in the ``off'' state. The activity is low because the interaction domain reduces the rate of the breakdown of the thioester intermediate which accumulates and therefore prevents the enzyme from embarking on another round of catalysis.


Figure 10: Schematic representation of carbamyl phosphate synthetase. The diagram is a schematic representation of the proposed model for the linkage between the GLNase and CPSase domains. In the absence of ATP and bicarbonate, the glutaminase domain has low activity (off state). The CPSase domain interacts with both GLNase subdomains although the interactions with the catalytic subdomain are weak and not sufficient to form a stable complex. The thioester forms and ammonia is released, but its breakdown is suppressed by the interaction subdomain. The thioester accumulates and glutamine hydrolysis is very slow. In the presence of ATP and bicarbonate (on state), a conformational change is induced within the CPSase domain which is then transmitted to the GLNase domain. The interaction domain no longer impedes the hydrolysis of the thioester. The active enzyme is regenerated for another round of catalysis, and ammonia is provided for carbamyl phosphate synthesis.



The binding of ATP and bicarbonate to the synthetase domain induces a conformational change which is transmitted to the interaction domain, with which it intimately interacts, and triggers the ``on'' state. These structural changes disrupt the attenuating interactions between the catalytic and interaction subdomains and relieves the inhibition. The thioester once formed rapidly breaks down and the enzyme turns over.

The mechanism by which the interaction domain suppresses the activity of the catalytic subdomain remains unknown. However, as we suggested previously(14) , the catalytic subdomain may in turn be comprised of two smaller structural units, since controlled proteolysis experiments demonstrated a hypersensitive cleavage site characteristic of an interdomain linker or hinge located in the middle (residue 287) of the subdomain. The two halves carry a very different charge so they may associate by electrostatic interactions. The calculated pI of the 12.8-kDa segment (residues 174-287) on the amino end is 9.7, while the 8.5-kDa segment (residues 288-365) on the carboxyl side has a pI of 4.1. It is interesting that the active site cysteine, Cys-252 is present in the amino half while the other 2 active site residues identified thus far in CAD, (^3)His-336 and Glu-338 are located in the carboxyl half of the catalytic subdomain. Moreover, there is a 10-residue, highly conserved sequence, region IV (residues 291-300), located in the carboxyl half of the catalytic domain immediately adjacent to the cleavage site. Since this sequence is conserved only in the CPSases, not in any other amidotransferases, we had suggested that region IV interacts specifically with the CPS synthetase domain and mediates interdomain signaling. In E. coli CPSase(49) , mutation of a region IV histidine residue (corresponding to His-295 in the CAD sequence) reduces glutamine binding. Moreover, saturating glutamine protects the catalytic subdomain from proteolysis, (^4)which supports the idea that this putative hinge is located near the glutamine binding site or undergoes a change in conformation when glutamine binds. An attractive idea, which remains to be tested, is that the interaction domain may alter the juxtaposition of the two halves of the catalytic subdomain and thus prevents the active site from fully forming or assuming the optimal conformation for catalysis.


FOOTNOTES

*
This research was supported by United States Public Health Service Grant GM47399. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: CPSase, carbamyl phosphate synthetase; GLNase, glutaminase activity or domain; CAD, polypeptide having CPSase, aspartate transcarbamylase, and dihydroorotase activities; kb, kilobase pair(s); bp, base pair(s); HPLC, high performance liquid chromatography; DTT, dithiothreitol.

(^2)
The only exception is the mammalian mitochondrial CPSase I which catalyzes the first step of the urea cycle and uses ammonia as a nitrogen donor(4) .

(^3)
H. I. Guy, A. Hewagama, and D. R. Evans, unpublished results.

(^4)
H. Kim, unpublished observations.


ACKNOWLEDGEMENTS

We wish to thank Jay Binder for expert technical assistance and Dr. Carol Lusty (The Public Health Research Institute of the City of New York) and Dr. Evan Kantrowitz (Boston College, Chestnut Hill, MA) for their generous gifts of plasmids and strains.


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