INTRODUCTION

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The nucleotide CTP is required for the synthesis of RNA, DNA, phospholipids, and sialoglycoproteins (1). CTP is synthesized from UTP via the reaction catalyzed by the cytosolic-associated enzyme CTP synthetase (EC 6.3.4.2, UTP: ammonia ligase (ADP-forming)) (2, 3) (Fig. 1). In eukaryotic cells, the regulation of CTP synthetase activity plays an important role in the balance of nucleotide pools (4-9) and in the synthesis of membrane phospholipids (10, 11). Moreover, unregulated CTP synthetase activity is a common property of leukemic cells (12) and rapidly growing tumors found in liver (13), colon (14), and lung (15). Overall, these observations emphasize the importance of studies to understand the regulation of CTP synthetase activity.

Our laboratory utilizes the yeast Saccharomyces cerevisiae as a model eukaryote to study the regulation of CTP synthetase activity. The yeast enzyme is encoded by the URA7 (7) and URA8 (8) genes. The URA7- (9) and URA8-encoded (16) CTP synthetases have been purified to apparent homogeneity and characterized with respect to their enzymological and kinetic properties. These CTP synthetases exhibit positive cooperative kinetics with respect to UTP and ATP (9, 16). This cooperativity has been attributed to the nucleotide-dependent oligomerization of the dimeric form of the enzyme to the tetrameric form (9, 16). Neither the URA7- nor the URA8-encoded CTP synthetase is essential provided that cells possess one functional enzyme (7, 8). Phenotypic analysis of ura7 and ura8 mutants (8) and the biochemical characterization of the URA7- and URA8-encoded enzymes (9, 16) have shown that the two CTP synthetases are not functionally identical. Moreover, the URA7-encoded enzyme is more abundant than the URA8-encoded enzyme (11) and is responsible for the majority of the CTP synthesized in vivo (8).

URA7-encoded CTP synthetase activity is regulated by CTP product inhibition (9) and by phosphorylation via protein kinase A (17) and by protein kinase C (18, 19). The inhibition of CTP synthetase activity by CTP regulates the cellular concentration of CTP in growing cells (9, 11). This regulation plays a role in the synthesis of phosphatidylcholine, the major membrane phospholipid in S. cerevisiae (11). Phosphorylation of the purified URA7-encoded CTP synthetase by protein kinase A (17) and by protein kinase C (18, 19) results in the stimulation of activity. The mechanism of stimulation by each of these protein kinases is the same and includes an increase in the V_max, a decrease in the K_m for ATP, and a decrease in the positive cooperative kinetics of the enzyme with respect to ATP (17, 19). The phosphorylation of the purified CTP synthetase by protein kinase A and protein kinase C also causes a decrease in the sensitivity of the enzyme to inhibition by CTP (17, 19). Although the effects of both protein kinases on CTP synthetase are the same, phosphopeptide mapping experiments have indicated that the protein kinase A and protein kinase C sites on the enzyme differ (17, 18). The deduced amino acid sequence of the URA7-encoded CTP synthetase has one potential target site for protein kinase A and eight potential target sites for protein kinase C. Results of labeling experiments under growth conditions that regulate the activities of protein kinase A and protein kinase C indicate that the phosphorylation of CTP synthetase is mediated by these protein kinases in vivo (17, 19).

Ligand-induced oligomerization of an enzyme is a major mechanism for the regulation of its activity (20). In this work, we examined the requirements and the kinetics of the ligand-induced tetramerization of the URA7-encoded CTP synthetase. Our studies also revealed that the phosphorylation of CTP synthetase by protein kinase A and protein kinase C played a significant role in the tetramerization and activation of the enzyme.

	EXPERIMENTAL PROCEDURES

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Materials-- Nucleotides, adenylyl imidodiphosphate (AMP-PNP),¹ L-glutamine, alkaline phosphatase-agarose, molecular mass standards for gel filtration chromatography, and bovine serum albumin were purchased from Sigma. Protein kinase A catalytic subunit (bovine heart) and protein kinase C (rat brain) were purchased from Promega. Protein assay reagent and molecular mass standards for SDS-polyacrylamide gel electrophoresis were purchased from Bio-Rad. Reagents for electrophoresis were purchased from National Diagnostics. Superose 6 was purchased from Amersham Pharmacia Biotech.

Purification of CTP Synthetase-- URA7-encoded CTP synthetase was purified to homogeneity by ammonium sulfate fractionation of the cytosol followed by chromatography with Sephacryl 300 HR, Q-Sepharose, Affi-Gel Blue, and Superose 6 (9). The specific activity of the pure enzyme was 2.5 µmol/min/mg.

Phosphorylation and Dephosphorylation of Purified CTP Synthetase-- Purified CTP synthetase was phosphorylated with protein kinase A as described previously (17) using the bovine heart catalytic subunit. The bovine heart protein kinase A catalytic subunit is structurally and functionally identical to the S. cerevisiae protein kinase A catalytic subunit (21). CTP synthetase was phosphorylated with protein kinase C as described previously (18) using a rat brain preparation of the enzyme. Rat brain protein kinase C phosphorylates and activates CTP synthetase in the same manner as that of purified protein kinase C isolated from S. cerevisiae (19). The protein kinase A and protein kinase C preparations used in our studies were judged to be essentially pure as determined by SDS-polyacrylamide gel electrophoresis. Native purified CTP synthetase was dephosphorylated with alkaline phosphatase attached to beaded agarose (18).

Superose 6 Gel Filtration Chromatography-- A Superose 6 column (1 × 24 cm) attached to a Pharmacia FPLC system was equilibrated and eluted with 50 mM Tris-HCl (pH 8.0), 2 mM L-glutamine, 10 mM 2-mercaptoethanol, 10 mM MgCl₂, and 0.1 mM GTP in the absence and presence of the indicated concentrations of ATP and UTP at 5 °C. The column was calibrated with blue dextran 2000 (for the void volume), thyroglobulin (669 kDa), apoferritin (443 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), and carbonic anhydrase (29 kDa). Purified CTP synthetase was incubated in the Superose 6 chromatography buffers (0.1 ml total volume) for 5 min and then applied and eluted from the Superose 6 column at a flow rate of 15 ml/h. Fractions (0.47 ml) were collected and analyzed for CTP synthetase protein by SDS-polyacrylamide gel electrophoresis.

Electrophoresis and Analysis of CTP Synthetase Protein-- SDS-polyacrylamide gel electrophoresis (22) was performed with 10% slab gels. Molecular mass standards were phosphorylase b (92.5 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), and soybean trypsin inhibitor (21.5 kDa). Proteins on SDS-polyacrylamide gels were stained with silver (23). The density of the CTP synthetase bands on SDS-polyacrylamide gels was quantified by scanning densitometry.

Enzyme Assays, Protein Determination, and Analysis of Kinetic Data-- CTP synthetase activity was determined by measuring the conversion of UTP to CTP (molar extinction coefficients of 182 and 1520 M¹ cm¹, respectively) by following the increase in absorbance at 291 nm on a recording spectrophotometer (3). The standard reaction mixture contained 50 mM Tris-HCl (pH 8.0), 2 mM UTP, 2 mM ATP, 2 mM L-glutamine, 0.1 mM GTP, 10 mM MgCl₂, 10 mM 2-mercaptoethanol, and an appropriate dilution of enzyme protein in a total volume of 0.1 ml. Enzyme assays were performed in triplicate with an average standard deviation of ±3%. All assays were linear with time and protein concentration. A unit of enzyme activity was defined as the amount of enzyme that catalyzed the formation of 1 µmol of product/min. Protein was determined by the method of Bradford (24) using bovine serum albumin as the standard. Kinetic data were analyzed according to the Michaelis-Menten and Hill equations using the EZ-FIT Enzyme Kinetic Model Fitting Program (25).

	RESULTS

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Effects of Substrates and Cofactors on the Tetramerization of CTP Synthetase-- The oligomeric forms of CTP synthetase were analyzed by Superose 6 gel filtration chromatography followed by the quantification of column fractions by scanning densitometry of silver-stained SDS-polyacrylamide gels. In contrast to measuring activity (9), the measurement of CTP synthetase protein permitted the identification of inactive oligomeric forms of the enzyme. We examined the effects of the substrates and cofactors used for the CTP synthetase reaction (Fig. 1) on the tetramerization of the purified native enzyme. CTP synthetase catalyzes the conversion of UTP to CTP in two sequential steps (26, 27). In the first step, UTP is phosphorylated with ATP to form 4-phospho-UTP via an ATPase reaction. The second step involves a glutaminase reaction where glutamine is converted to glutamate and NH₃. The NH₃ generated in the glutaminase reaction combines with the phosphorylated UTP to form CTP. GTP activates the reaction by accelerating the formation of a covalent glutaminyl enzyme catalytic intermediate (3, 28). Mg²⁺ ions are required to form metal ion-nucleotide complexes during the enzyme reaction (29). In experiments to examine the dependence of specific reaction components on enzyme tetramerization, each of the other reaction components were present at saturating concentrations.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Reaction catalyzed by CTP synthetase. The figure shows the structures of UTP and CTP and the reaction catalyzed by CTP synthetase.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Elution profiles of the CTP synthetase protein after chromatography with Superose 6 in the absence and presence of subsaturating and saturating concentrations of ATP and UTP. Purified CTP synthetase (50 µg) was subjected to Superose 6 chromatography in the absence (panel A) and presence (panels B and C) of the indicated concentrations of ATP and UTP as described under "Experimental Procedures." The concentrations of glutamine, GTP, and MgCl2 were 2, 0.1, and 10 mM, respectively. The column was calibrated with blue dextran 2000 (for the void volume), thyroglobulin (669 kDa), apoferritin (443 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), and cytochrome c (12.4 kDa). Fractions (0.47 ml) were collected, and the relative amount of the CTP synthetase protein in the column fractions were quantified by SDS-polyacrylamide gel electrophoresis and densitometry of silver-stained gels. The positions of the dimeric and tetrameric CTP synthetase oligomers are indicated in the figure. The inset in panel B shows a portion of the SDS-polyacrylamide gel that was used for the analysis of the CTP synthetase protein in the column fractions.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   The effects of substrates and cofactors on the tetramerization of CTP synthetase. Purified CTP synthetase (50 µg) was subjected to Superose 6 chromatography in the absence and presence of 2 mM ATP, 2 mM UTP, 2 mM glutamine (Gln), 0.1 mM GTP, and 10 mM MgCl2 as described under "Experimental Procedures." The oligomeric forms of CTP synthetase were quantified as described in the legend to Fig. 2.

Dependence of Tetramerization on the Concentrations of ATP and UTP-- URA7-encoded CTP synthetase activity exhibits positive cooperative kinetics with respect to ATP and UTP when measured with subsaturating and saturating concentrations of UTP and ATP, respectively (9, 17, 19). This cooperative kinetic behavior is presumably because of the nucleotide-dependent tetramerization of the enzyme (9). To further explore this hypothesis, we examined the dependence of CTP synthetase tetramerization on ATP and UTP. In these experiments, the concentrations of glutamine, GTP, and Mg²⁺ ions were maintained at saturating concentrations of 2 mM, 0.1 mM, and 10 mM, respectively. At subsaturating (0.1 mM) and saturating (2 mM) concentrations of UTP, the tetramerization of the CTP synthetase protein was dependent on ATP in a dose-dependent manner (Fig. 4, A and B, respectively). At 0.1 mM UTP the maximum amount (60%) of tetramer formed was at 1.2 mM ATP (Fig. 4A), whereas at 2 mM UTP the maximum amount (93%) of tetramer formed was at 0.4 mM ATP (Fig. 4B). The kinetics of tetramerization correlated in general with the kinetics of CTP synthetase activity (9, 17, 19). The V_max and K_m values with respect to ATP at 0.1 mM UTP are 2.11 units/mg and 1.44 mM, respectively (17). The V_max and K_m values with respect to ATP at 2 mM UTP are 2.5 units/mg and 0.85 mM, respectively (17). Similarly, at subsaturating (0.5 mM) and saturating (2 mM) concentrations of ATP, the tetramerization of CTP synthetase was dependent on UTP in a dose-dependent manner (Fig. 4, C and D, respectively). Moreover, tetramerization of the enzyme was absolutely dependent on UTP. At both subsaturating and saturating concentrations of ATP, maximum tetramerization was obtained at 0.3 mM UTP. However, the maximum amount of tetramer present at 2 mM ATP was 92%, whereas the maximum amount of tetramer present at 0.5 mM ATP was 50%. The amounts of the tetramer formed in these tetramerization experiments correlated in general with the kinetics of CTP synthetase activity (17). The V_max and K_m values with respect to UTP at 0.5 mM ATP are 0.76 units/mg and 0.08 mM, respectively (17). The V_max and K_m values with respect to UTP at 2 mM ATP are 2.3 units/mg and 0.05 mM, respectively (17).

View larger version (38K): [in this window] [in a new window]   Fig. 4.   Dependence of CTP synthetase tetramerization on the concentrations of ATP and UTP. Purified CTP synthetase (50 µg) was subjected to Superose 6 chromatography in the presence of the indicated concentrations of ATP using 0.1 mM UTP (panel A) and 2 mM UTP (panel B), and the indicated concentrations of UTP using 0.5 mM ATP (panel C) and 2 mM ATP (panel D) as described under "Experimental Procedures." The oligomeric forms of CTP synthetase were quantified as described in the legend to Fig. 2. The concentrations of glutamine, GTP, and MgCl2 were 2, 0.1, and 10 mM, respectively.

Effect of AMP-PNP on the Tetramerization of CTP Synthetase-- As indicated above, ATP facilitated the UTP-dependent tetramerization of CTP synthetase. We next questioned if the phosphorylation of UTP by ATP was required for this process. For this experiment, we used the nonphosphorylating ATP analogue AMP-PNP that is commonly used to inhibit ATPase reactions (30). Initial studies were directed toward showing that AMP-PNP was a competitive inhibitor of CTP synthetase activity with respect to ATP. In our kinetic experiments, the dependence of CTP synthetase activity on ATP was measured at a saturating concentration of UTP (2 mM). CTP synthetase activity was measured with respect to ATP in the absence and presence of AMP-PNP (Fig. 5A). AMP-PNP inhibited CTP synthetase activity in a dose-dependent manner at each ATP concentration. AMP-PNP also caused an increase in the positive cooperative kinetic behavior of CTP synthetase activity with respect to ATP with Hill numbers increasing from 1.0 to 1.4. Analysis of the kinetic data according to the Hill equation showed that AMP-PNP caused an increase in the apparent K_m values (from 0.2 to 0.8 mM) for ATP but had little effect on the apparent V_max values. These results were consistent with AMP-PNP being a competitive inhibitor (K_i = 0.5 mM) with respect to ATP (31). We examined if 2 mM AMP-PNP could serve as a substrate for CTP synthetase using saturating concentrations of UTP, glutamine, GTP, and Mg²⁺ ions. AMP-PNP did not serve as a substrate. These data indicated that AMP-PNP inhibited CTP synthetase activity by competing with the ATP-dependent phosphorylation of UTP.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   The effect of AMP-PNP on the kinetics of CTP synthetase activity with respect to ATP and on the tetramerization of CTP synthetase. Panel A, CTP synthetase activity was measured as a function of the concentration of ATP at set AMP-PNP concentrations of 0 mM (), 0.5 mM (), 1 mM (), and 2 mM (). Panel B, purified CTP synthetase (50 µg) was subjected to Superose 6 chromatography in the presence of the indicated concentrations of ATP and AMP-PNP as described under "Experimental Procedures." The oligomeric forms of CTP synthetase were quantified as described in the legend to Fig. 2. The concentrations of UTP, glutamine, GTP, and MgCl2 were 2, 2, 0.1, and 10 mM, respectively.

Effect of CTP on the Tetramerization of CTP Synthetase-- URA7-encoded CTP synthetase activity is potently inhibited by its product CTP (9, 17, 19). However, as described previously (9), CTP (2 mM) did not inhibit the ATP/UTP-dependent tetramerization of the enzyme (data not shown). Thus, the inhibition of activity by CTP does not occur by a mechanism that prevents enzyme tetramerization (9) and that the tetrameric enzyme is regulated by CTP. We also examined the effect of CTP on the tetramerization of the enzyme in the absence of UTP using a subsaturating concentration of ATP. At a concentration of 2 mM, CTP completely substituted for UTP in the ability to promote enzyme tetramerization (Fig. 6). In fact, 0.1 mM CTP promoted the tetramerization of the enzyme more effectively than 0.1 mM UTP (Fig. 6). However, CTP synthetase activity was not required for enzyme tetramerization. As discussed above, most of the enzyme (95%) existed as a tetramer in the presence of saturating concentrations of ATP and UTP in the absence of glutamine and GTP (Fig. 3). Moreover, CTP synthetase existed as a tetramer in the presence of UTP in the absence of ATP (Fig. 4B). Under both of these conditions, there is no CTP synthetase activity. CTP may substitute for UTP in promoting enzyme tetramerization because CTP is similar in structure to UTP. CTP contains an amino group at the C-4 position of the pyrimidine ring, whereas UTP contains a carbonyl group (Fig. 1).

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Effect of CTP on the tetramerization of CTP synthetase. Purified CTP synthetase (50 µg) was subjected to Superose 6 chromatography in the presence of the indicated concentrations of UTP and CTP as described under "Experimental Procedures." The oligomeric forms of CTP synthetase were quantified as described in the legend to Fig. 2. The concentrations of ATP, glutamine, GTP, and MgCl2 were 0.5, 2, 0.1, and 10 mM, respectively.

Effect of Phosphorylation on the Tetramerization of CTP Synthetase-- Phosphorylation of the native purified CTP synthetase by protein kinase A (17) and protein kinase C (18, 19) results in the stimulation of CTP synthetase activity through a common mechanism that affects the kinetic properties of the enzyme. The effects of phosphorylation on the kinetics of CTP synthetase activity are most pronounced at subsaturating concentrations of ATP and UTP (17, 19). For example, at 0.1 mM UTP, the apparent K_m value for ATP for the native enzyme is 1.5 mM, whereas the apparent K_m values for the enzyme phosphorylated with protein kinase A (17) and protein kinase C (19) are 0.9 mM and 0.6 mM, respectively. Given the fact that phosphorylation affects the activity and kinetic behavior of CTP synthetase activity with respect to ATP and UTP, we explored the hypothesis that phosphorylation of the enzyme plays a role in the nucleotide-dependent tetramerization of the enzyme. As discussed previously (17), the CTP synthetase that we purified was already partially phosphorylated (18). Thus, the tetramerization experiments described here on the native purified enzyme reflected the property of this phosphorylated state of the enzyme. We could not utilize dephosphorylated CTP synthetase in these studies because the dephosphorylated enzyme was inactive at subsaturating concentrations of ATP and UTP. The native purified enzyme was phosphorylated with protein kinase A and protein kinase C. The phosphorylated enzyme preparations were then subjected to gel filtration chromatography in the presence of subsaturating concentrations (0.4 mM and 0.8 mM) of ATP using a subsaturating concentration (0.1 mM) of UTP. By using subsaturating concentrations of ATP and UTP, we could more readily observe the effects of phosphorylation on the tetramerization of the enzyme. In addition, nearly all of the native enzyme existed as a tetramer in the presence of saturating concentrations of ATP and UTP (Fig. 2). The amounts of tetramer of the protein kinase A- and protein kinase C-phosphorylated forms of CTP synthetase were greater at the two subsaturating ATP concentrations than the amounts of tetramer of the native enzyme (Fig. 7). In other words, phosphorylation of the enzyme facilitated the formation of the tetramer at lower concentrations of ATP.

View larger version (31K): [in this window] [in a new window]   Fig. 7.   Effect of phosphorylation of CTP synthetase by protein kinase A and protein kinase C on the tetramerization of CTP synthetase. CTP synthetase was phosphorylated with protein kinase A (PKA) and with protein kinase C (PKC) as described under "Experimental Procedures." Native and phosphorylated CTP synthetase (50 µg) were subjected to Superose 6 chromatography in the presence of the indicated concentrations of ATP using 0.1 mM UTP. The oligomeric forms of CTP synthetase were quantified as described in the legend to Fig. 2. The concentrations of glutamine, GTP, and MgCl2 were 2, 0.1, and 10 mM, respectively.

Effect of Dephosphorylation of CTP Synthetase on Tetramerization-- As discussed above, the dephosphorylation of the native purified CTP synthetase with alkaline phosphatase results in the inactivation of activity (17, 18). Rephosphorylation of the alkaline phosphatase-treated enzyme with protein kinase A and protein kinase C results in a partial restoration in CTP synthetase activity (17, 18). We questioned if the dephosphorylation of CTP synthetase affected the oligomeric structure of the enzyme. When the native CTP synthetase was dephosphorylated with alkaline phosphatase and then examined for its oligomeric structure in the presence of saturating concentrations of ATP and UTP, almost all (95%) of the enzyme was found in its dimeric form (Fig. 8). The alkaline phosphatase-treated enzyme was rephosphorylated with protein kinase A and protein kinase C and then examined for its oligomeric structure in the presence of 2 mM ATP and 2 mM UTP. About 30 and 40% of the enzyme rephosphorylated with protein kinase A and protein kinase C, respectively, existed as a tetramer (Fig. 8). The alkaline phosphatase-treated CTP synthetase was also rephosphorylated with a combination of protein kinase A and protein kinase C, and then examined for the oligomeric structure in the presence of ATP and UTP. Under these conditions, about 60% of the rephosphorylated enzyme existed as a tetramer (Fig. 8). The extent of the nucleotide-dependent tetramerization of the CTP synthetase rephosphorylated with protein kinase A and protein kinase C correlates in general with the extent of the CTP synthetase activity that is recovered after rephosphorylation with these protein kinases (17).

View larger version (27K): [in this window] [in a new window]   Fig. 8.   Effect of dephosphorylation and rephosphorylation on the tetramerization of CTP synthetase. CTP synthetase was dephosphorylated by treatment with alkaline phosphatase. Samples of the alkaline phosphatase-treated (AP-treated) enzyme were then phosphorylated with protein kinase A (PKA), protein kinase C (PKC), and a combination of protein kinases A and C (PKA + PKC) as described under "Experimental Procedures." The indicated enzyme preparations were subjected to Superose 6 chromatography in the presence of 2 mM ATP and 2 mM UTP. The oligomeric forms of CTP synthetase were quantified as described in the legend to Fig. 2. The concentrations of glutamine, GTP, and MgCl2 were 2, 0.1, and 10 mM, respectively.

	DISCUSSION

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The roles of ATP and UTP in the CTP synthetase reaction are complex. These nucleotides serve as substrates in the reaction, and they are responsible for the tetramerization of the enzyme. UTP was essential for enzyme tetramerization and ATP facilitated the UTP-dependent tetramerization through its role in the phosphorylation of UTP. Whereas this step in the overall CTP synthetase reaction was required for maximum tetramerization of the enzyme, the glutaminase step was not required. There was a correlation between the kinetics of tetramerization and the kinetics of CTP synthetase activity. This was consistent with the conclusion that tetramerization was required for enzyme activity and that the cooperative kinetics with respect to ATP and UTP was a reflection of the tetramerization of the enzyme. However, catalysis was not required for tetramerization. Our studies also showed that CTP, a potent inhibitor of CTP synthetase activity (9), did not inhibit enzyme tetramerization. Thus, CTP inhibits the activity of CTP synthetase when the enzyme is in its tetrameric form.

Given the fact that phosphorylation of CTP synthetase by protein kinase A and protein kinase C stimulated CTP synthetase activity by a mechanism that affected the kinetics of activity with respect to ATP (17, 19), we examined the hypothesis that phosphorylation facilitated the nucleotide-dependent tetramerization of the enzyme. The phosphorylation of the native purified enzyme facilitated the nucleotide-dependent tetramerization of the enzyme. ATP and UTP did not induce tetramerization when the enzyme was dephosphorylated with alkaline phosphatase. Moreover, the dephosphorylated enzyme was inactive. These results indicated that the phosphorylation of the enzyme was required for nucleotide-dependent tetramerization and activity. The inactive enzyme was presumably because of CTP synthetase remaining in the dimeric form, which could no longer be induced to form a tetramer in the presence of ATP and UTP. Phosphorylation by itself did not induce tetramerization. The native enzyme preparation contains a population of phosphorylated enzyme (18) and essentially all of the native enzyme was dimeric in the absence of ATP and UTP. Phosphorylation of the alkaline phosphatase-treated CTP synthetase by protein kinase A and protein kinase C resulted in a partial restoration in the ability of the enzyme to form a tetramer in the presence of ATP and UTP. Moreover, the effects of protein kinase A and protein kinase C on enzyme tetramerization were not additive nor synergistic. This tetramerization is accompanied by a partial activation of CTP synthetase activity (17, 18). The partial activation of enzyme activity was presumably because of the population of enzyme that formed a tetramer after rephosphorylation. Full tetramerization and activation of CTP synthetase by protein kinase A and protein kinase C may require additional protein kinase phosphorylations and/or a hierarchical phosphorylation sequence may exist (32). It is worth noting that the deduced amino acid sequence of the URA7-encoded CTP synthetase contains eight potential phosphorylation sites for casein kinase II. Additional studies will be required to address these hypotheses.

The promotion of oligomerization by phosphorylation has been described for other proteins (33-37). A relevant example is the phosphorylation and tetramerization of mammalian glycogen phosphorylase. Glycogen phosphorylase, an allosterically regulated enzyme, is inactive in the dephosphorylated state and is activated after phosphorylation with phosphorylase kinase (1). Phosphorylation is accompanied by the oligomerization of dimers to tetramers and changes in the allosteric properties of the enzyme with respect to substrates, activators, and inhibitors (1). Crystal structure analysis of glycogen phosphorylase has shown that phosphorylation causes conformational changes within the dimeric enzyme that enables the formation of a tetramer through intersubunit interactions (35, 37). Structural analysis will be required to elucidate the molecular mechanism of phosphorylation-promoted tetramerization of CTP synthetase.

The tetramerization of the CTP synthetase from S. cerevisiae was similar to that reported for the CTP synthetase from Escherichia coli (38) with respect to its requirements of ATP and UTP for maximum tetramerization. However, there were differences between the yeast and bacterial enzymes for tetramerization. The tetramerization of the yeast enzyme was not induced by ATP in the absence of UTP, whereas the enzyme from E. coli forms a tetramer with ATP alone (38). The tetramerization of the yeast CTP synthetase was dependent on the phosphorylation of the enzyme, whereas the tetramerization of the E. coli enzyme has not been shown to be dependent on enzyme phosphorylation. Analysis of the deduced protein sequence of the pyrG gene encoding the E. coli CTP synthetase did not reveal any potential phosphorylation target sequences for the enzyme. The regulation of tetramerization and activity of the S. cerevisiae CTP synthetase by phosphorylation is a level of regulation that does not appear to exist in E. coli.

Based on the data presented in this study, we propose a working model for the role of phosphorylation in the tetramerization and activation of the yeast CTP synthetase (Fig. 9). This model was adapted from the model of Levitzki and Koshland (38) for the nucleotide-dependent tetramerization of the E. coli CTP synthetase. The enzyme exists as a dimer through the interaction of dimer binding domains on the monomeric enzyme. Dephosphorylated dimeric CTP synthetase (I) is inactive. Phosphorylation of inactive dimeric CTP synthetase results in a conformational change to form phosphorylated dimeric enzyme (II). The binding of UTP and ATP to the phosphorylated dimeric form of CTP synthetase induces the association of phosphorylated dimers to form the phosphorylated tetrameric enzyme (III). The ATP-dependent phosphorylation of UTP by the enzyme facilitates tetramerization. Phosphorylation stimulates activity by lowering the concentration of nucleotides required to facilitate the tetramerization of the enzyme. This occurs through a conformation change that increases the affinity of enzyme dimers for the nucleotide substrates.

View larger version (13K): [in this window] [in a new window]   Fig. 9.   Model for the role of phosphorylation in the nucleotide-dependent tetramerization and activation of CTP synthetase. The square represents the dephosphorylated CTP synthetase subunit, and the circle containing the letter P represents the phosphorylated enzyme subunit. Roman numerals in the figure indicate the different oligomeric forms of CTP synthetase discussed in the text.

The studies reported here on the nucleotide-dependent tetramerization of CTP synthetase advances our understanding of the mechanism of the enzyme reaction in vitro. The steady-state cellular concentrations of ATP (0.57-2.3 mM) and UTP (0.6 mM) (7, 11, 39) are within the range of the subsaturating to saturating concentrations of these nucleotides that regulate CTP synthetase activity in vitro (9). Indeed, the effects of phosphorylation on enzyme tetramerization were most dramatic when the concentrations of ATP and UTP were subsaturating. Thus, the tetramerization and stimulation of CTP synthetase activity by phosphorylation may be a mechanism by which the cell regulates CTP synthesis when cellular nucleotide levels are limiting. However, additional studies will be required before the physiological significance of the in vitro observations reported in this work is established.