polypeptide chains of 17,000 daltons. The six larger or regulatory site (Asp-19-+

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 266, No. 33, Issue of November 25, pp. 22154-22158,1391 Printed in U. S. A. The Synergistic Inhibition of Escherichia coli Aspartate Carbamoyltransferase by UTP in the Presence of CTP Is Due to the Binding of UTP to the Low Affinity CTP Sites* (Received for publication, March 20, 1991) Yang Zhang and Evan R. KantrowitzS From the Department of Chemistry, Boston College, Chestnut Hill,Massachusetts 02167 Escherichia coli aspartate carbamoyltransferase In the presence of CTP, UTP inhibits the enzyme more than controls pyrimidine biosynthesis by feedback inhibi- CTP alone (6), and as suggested by Wild et al. (6) the tion involving both CTP and UTP, although UTP only synergistic inhibition of aspartate carbamoyltransferase by inhibits the enzyme in the presence of CTP (Wild, J. both CTP and UTP provides a satisfying logic for ensuring a R., Loughrey-Chen, S. J., and Corder, T. S. (1989) balance of endogenous pyrimidine nucleotide pools. Proc. Natl. Acad. Sci. U. S. A. 86, 46-50). The mech- The E. coli aspartate carbamoyltransferase enzyme is peranism by which the enzyme can discriminate between haps the best studied allosteric enzyme both on the biochemthese two pyrimidines is unknown, as well as where ical and structural levels (7-16). The 310,000-dalton holoen- UTP binds and its mode of action. A mutant version of zyme consists of six polypeptide chains of 34,000 daltons and the enzyme with a single amino acid substitution in six the polypeptide chains of 17,000 daltons. The six larger or regulatory site (Asp-19-+ Ala) causes loss of the synergistic inhibition of UTP in the presence of CTP, and catalytic chains are grouped together into two trimers that furthermore, this enzyme is inhibited by UTP alone. contain the active sites shared across the interface between Analysis of CTP binding to the mutant enzyme reveals adjacent chains (17-21), while the six smaller or regulatory that UTP can bind to the mutant enzyme in the absence polypeptide chains are grouped into three dimers that contain of CTP but not in its presence. This is completely the binding sites for ATP and CTP. opposite to the wild-type enzyme in which case UTP Both CTP and ATP bind and compete for the same site on only exhibits significant binding in the presence of the six regulatory chains of the holoenzyme (22); furthermore, CTP. Further analysis of the binding data for wild- the these regulatory sites can be divided according to their affinity type enzyme reveals that, in the presence of UTP, CTP for the nucleotides. ATP and CTP bind to three of the sites only binds to three sites, although CTP binds to six with high affinity and to the other three sites with approxisites, three with high affinity and three with low affin- mately 10-fold lower affinity (23-27). Neither the number nor ity in the absence of UTP. Parallel UTP binding ex- location of the UTP-binding sites nor the mechanism of periments in the presence of CTP suggest that UTP inhibition of the enzyme by UTP has been determined. binds to the three weak CTP sites. The Asp-19 -., Ala In order to better understand the interactions of aspartate substitution prevents UTP binding in the presence of carbamoyltransferase with its regulatory nucleotides, we have CTP and allows UTP to bind and inhibit the enzyme previously in used site-specific mutagenesis to alter amino acid the absence of CTP. Since the x-ray data indicate no side chains involved in nucleotide binding (28, 29) as identispecific interactions between the amino group of cy- fied by x-ray crystallography (30-32). Here we report our tosine and amino acid side chains in the regulatory results on the analysis of a mutant enzyme with a substitution binding site, the discrimination between UTP and CTP of alanine at position 19 in the regulatory chain. The analysis by the wild-type enzyme must be due to subtle differof these data along with new data for the wild-type enzyme ences in the binding sites rather than direct side chain contacts. clearly establishes the binding site of UTP on the wild-type enzyme and begins to unravel its mode of action. EXPERIMENTAL PROCEDURES In Escherichia coli the enzyme aspartate carbamoyltransferase exerts control of pyrimidine biosynthesis by a combination of genetic, allosteric, and metabolic mechanisms. On the genetic level, the expression of the enzyme is regulated by an attenuation mechanism (1-3). In addition, the enzyme also exhibits sigmoidal allosteric kinetics for both of its substrates (4, 5), and the enzyme is metabolically regulated by both purines and pyrimidines. CTP, the end product of the pyrimidine pathway, inhibits the enzyme while ATP, the end product of the parallel purine pathway, activates the enzyme (5). * This work was supported by Grant GM26237 from the National Institute of General Medical Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked aduertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom all correspondence should be addressed. Materials Agar, ampicillin, L-aspartate, N-carbamoyl-L-aspartate, carbamoyl phosphate, potassium dihydrogen phosphate, ATP, CTP, UTP, and HEPES were purchased from Sigma. The carbamoyl phosphate was purified before use by precipitation from 50% (v/v) ethanol and stored desiccated at -20 C (5). Electrophoresis-grade acrylamide, agarose, urea, Tris, and enzyme-grade ammonium sulfate were obtained from ICN Biomedicals. As suggested by a reviewer, the inhibition of the enzyme by UTP in the presence of CTP should technically be called either concerted or multivalent. However, to be consistent with the previous literature concerning this phenomenon (6), we have chosen to use the word synergistic. Since UTP does inhibit the enzyme very slightly in the absence of CTP, synergistic inhibition may actually be more appropriate than either concerted or multivalent inhibition. The abbreviation used is: HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid. 22154

Methods Construction of the Asp-19r 3 Ala3 Mutation by Site-specific Mutagenesis-The substitution of alanine for Asp-19 of the regulatory chain of aspartate carbamoyltransferase was accomplished by sitespecific mutagenesis using the method of Zoller and Smith (33) with the modifications previously described (34,35). Single-stranded DNA from 15 candidates was isolated and sequenced by the dideoxy method (36). Two of the candidates gave the sequence corresponding to the mutation. A small fragment of the gene containing the mutation was removed with restriction enzymes and inserted into a plasmid which had the corresponding section of the wild-type gene removed (37). The mutation was verified a second time, after construction of the plasmid, employing single-stranded DNA copied from the plasmid using the helper phage M13K07 (38). Aspartate Caz$amoyltransferase Assay-The carbamoyltransferase activity was measured at 25 "C by either the colorimetric (39) or the ph-stat method (40). ph-stat assays were carried out with a Radiometer TTTBO titrator and an ABU80 autoburette. All colorimetric assays were performed in duplicate, and the data points shown in the figures are the average. Binding Measurements-The binding of CTP or UTP to the wildtype and the Asp-19r + Ala enzymes were determined by the technique of equilibrium dialysis using Spectra/Pro-2 (Spectrum Medical Industries) dialysis tubing. Dialysis experiments were carried out in microdialysis cells which hold 50 pl on each side of the dialysis membrane, which was pretreated as previously described (41). After equilibation for 18-20 h at 25 "C, 25-pI samples were removed from each side of the dialysis cell, and the concentration of UTP or CTP was determined by liquid scintillation employing a LKB 1217 Rackbeta liquid scintillation counter. Complete equilibration was confirmed under the experimental conditions. Equilibrium dialysis experiments were performed in 0.1 M HEPES, 0.2 mm EDTA, 2 mm 2- mercaptoethanol, ph 7, and the enzyme was dialyzed into this buffer before use. For the dialysis experiments in which the competition between ['HICTP and UTP was investigated, the enzyme and [3H]CTP concentrations were kept constant at 17 mg/ml and 0.24 mm (0.01 mci/ pl), respectively, on one side of the membrane while the concentration of UTP was varied on the other. Carbamoyl phosphate at an equilibrium concentration of 19.2 mm was also present in order to prevent the binding of the nucleotide effectors at the active site (42). Data Analysis-The analysis of the steady-state kinetic data was carried out as previously described by Silver et al. (43). The analysis of the structural data, based on the three-dimensional coordinates of the CTP-enzyme complex (30), was accomplished using the program QUANTA (Polygen Corp., Waltham, MA) on an IBM RISC/6000 computer. Other Methods-Oligonucleotide synthesis, enzyme purification, and determination of protein concentration were as previously described (37). RESULTS Binding of UTPICTP to Aspartate Carbamoyltransferase 22155 Kinetic Comparison of the Mutant and Wild-type Enzymes-Asp-19 of the regulatory chain (A~p-19r)~ residue measured at increasing concentrations of nucleotide (NTP). When UTP and CTP were present simultaneously, the abscissa corresponds located in the nucleotide-binding site that interacts with the to the total nucleotide concentration. A, effect of CTP alone (0) on ribose 3"OH group of ATP and CTP, the eamino group of the wild-type enzyme and the synergistic effect of CTP plus UTP Lys-56r, and the amide of His-20r (Fig. 1). Not unexpectantly, (0). When the concentration of CTP reached 2 mm, UTP was added most of the basic kinetic properties of the Asp-19r + Ala to the wild-type enzyme. The UTP concentration was increased from enzyme are very similar to those of the wild-type enzyme 0 to 2 mm while the concentration of CTP remained constant (2 including maximal velocity and substrate concentrations at mm). B, identical experiment as depicted in A for the Asp-19r + Ala enzyme. 0, CTP alone; half the maximal observed specific activity. However, UTP 0, CTP plus UTP. influences the activity of the mutant enzyme differently than the wild type. For the wild-type enzyme, the addition of UTP to the enzyme saturated with CTP causes a further decrease in activity (see Ref. 6 and Fig. 2A). For the Asp-19r +. Ala The notation used to name the mutant enzymes is, for example, the Asp-19r + Ala enzyme. The wild-type amino acid and location within the catalytic (c) or regulatory (r) chain is indicated to the left of the arrow while the new amino acid is indicated to the right of the arrow. The letter after the residue number indicates whether the location is in the catalytic (c) or regulatory (r) chain of aspartate carbamoyltransferase. FIG. 1. An a-carbon trace of the allosteric domain of one regulatory chain of wild-type aspartate carbamoyltransferase shown with CTP bound. ATP and CTP have been shown to bind and directly compete for the same site on the regulatory chain of the enzyme. Asp-19 of the regulatory chain interacts directly with the ribose 3'-OH of both ATP and CTP. Asp-19r also forms links to the 6-amino group of Lys-56 and the backbone amide of His-20 of the same regulatory chain. The three-dimensional coordinates of the enzyme used to produce this diagram were provided by W. N. Lipscomb. 1.01 FIG. 2. Influence of CTP and UTP on the carbamoyltransferase activity of the wild-type and the Asp-19r Ala enzymes. The activity of the wild-type and the Asp-19r + Ala enzymes were measured at 25 "C by the colorimetric method (39). Each data point was determined in duplicate, and the data points shown are the average. Measurements were made at ph 7.0 in 0.1 M imidazole acetate buffer at a saturating concentration of carbamoyl phosphate (19.2 mm) and 5 mm aspartate. Percentage relative activity was enzyme, UTP is not able to enhance the CTP inhibition (Fig. 2B). Even when the UTP concentration is increased to 25 mm, UTP does not synergistically inhibit the Asp-19r + Ala enzyme (data not shown). The loss of the synergistic inhibition by UTP could be the result of either a drastic decrease in UTP binding affinity or the inability of UTP, once bound, to inhibit the mutant enzyme. UTP also has a different affect on the Asp-19r + Ala than the wild-type enzyme in the absence of CTP. For the wild-type enzyme, in the absence of CTP, UTP causes only a very slight inhibition of the enzyme (Fig. 3); however, for the Asp-19r + Ala enzyme UTP signif-

22156 Binding of UTPICTP to Aspartate Carbamoyltransferase l " " " " ' 1 0 4 8 12 16 20 [UTP], mm FIG. 3. Influence of UTP on the activity of the wild-type (0) and the Asp-l9r 4 Ala (0) enzymes. Experimental conditions and procedures are identical to those described in Fig. 2. 2 3) 1 t I O 0 0 0 u '0 2 4 6 8 IO 12 [UTP), mm FIG. 4. Influence of UTP on the binding of CTP to wildtype aspartate carbamoyltransferase. The binding of CTP, RCTp, the moles of CTP bound per mol of aspartate carbamoyltransferase (M, 310,000), was measured by equilibrium dialysis as a function of [UTP]. Equilibrium dialysis experiments were performed in 0.1 M HEPES, 2.0 mm 2-mercaptoethanol, 0.2 mm EDTA buffer, ph 7.0. Carbamoyl phosphate was present at an equilibrium concentration of 20 mm to prevent nucleotide binding at the active site (42). icantly inhibits the enzyme even in the absence of CTP (Fig. 3). At least in the absence of CTP, these data indicate that UTP can bind to the mutant enzyme. UTP Enhances the Binding of CTP for the Wild Type but Not the Mutant Enzyme-In order to better understand the altered effects of UTP on the Asp-19r + Ala enzyme, we decided to first determine if UTP and CTP compete for the same site on the wild-type enzyme. As seen in Fig. 4, the binding of CTP, as measured by equilibrium dialysis, does not decrease as the concentration of UTP increases, as would be expected for a direct competition, but rather increases. The enhanced binding of CTP in the presence of UTP could be the result of either additional CTP sites becoming accessible, or UTP binding enhances the binding affinity of CTP. Kinetic data of the CTP/UTP synergism favor the latter (6). In order to distinguish between the above two possibilities, CTP binding to the wild-type enzyme was measured by equilibrium dialysis in the absence or presence of UTP. As seen in Fig. 5 and as reported previously (42), the binding of CTP alone to the wild-type enzyme is biphasic corresponding to two classes of sites having different affinity for CTP. When this experiment is repeated in the presence of 10 mm UTP two alterations to the CTP binding occur. First, the dissociation constant (KD) of CTP to the high affinity class of sites decreases substantially from 8.3 X to 3.6 X in the presence of UTP. Second, the KD for CTP binding to the low affinity sites increases significantly. In fact, in the presence of UTP, the affinity of the enzyme for CTP at the low affinity sites is so low that by equilibrium dialysis it cannot be determined if there is any CTP binding to this class of sites. A similar experiment was performed previously (6) over a very limited range of RCTp that was insufficient to detect the low affinity sites. Under identical conditions the binding of CTP was also measured for the Asp-19r + Ala enzyme. For the mutant enzyme the results were significantly different, and the binding of CTP to the enzyme is the same in the absence or presence of UTP and is almost identical to the 0 0 1 2 3 4 5 6 RNTP FIG. 5. Binding of CTP and UTP to the wild-type and Asp- 19r + Ala enzymes. Equilibrium dialysis experiments were performed in 0.1 M HEPES, 2.0 mm 2-mercaptoethanol, 0.2 mm EDTA buffer, ph 7.0, following the protocol previously reported (29). The binding data are represented as a Scatchard plot (47), RNW/[NTP]~ uersus RNTP, where RNTp is the number of moles of nucleotide bound per mole of aspartate carbamoyltransferase (M, 310,000) and [NTP]r is the concentration of free nucleotide. The binding experiments were carried out in the presence of an equilibrium concentration of 20 mm carbamoyl phosphate to prevent nucleotide binding at the active site (42). The binding of CTP was measured at 25 "C after an 18-20-h equilibrium time to the wild-type enzyme in the absence of UTP (0) and in the presence of an equilibrium concentration of 10 mm UTP (0). Under the same conditions, the binding of CTP to the Asp-19r -+ Ala enzyme was also measured in the absence of UTP (0) and in the presence of an equilibrium concentration of 10 mm UTP (m). The binding of UTP to the wild-type enzyme was determined at 25 "C (0) in the presence of an equilibrium concentration of 2 mm CTP. The binding of UTP to the wild-type (A) and the Asp- 19r + Ala (A) enzymes was also determined at 4 "C because the binding of UTP could not be detected at 25 "C. It has been reported the CTP and ATP bind six times more strongly at this temperature (24). binding of CTP alone to the wild-type enzyme, exhibiting both high and low affinity sites. These data suggest that either UTP is not binding to the mutant enzyme in the presence of CTP, with detectable affinity, or if UTP is binding, it can no longer induce an alteration in the binding of CTP. The Affinity of UTP in the Absence of GTP Is Higher for the Mutant Than the Wild-type Enzyme-The affinity of the wild-type enzyme for UTP, either in the presence or absence of CTP, is not high enough to measure by equilibrium dialysis at 25 "C. Since ATP and CTP have approximately 6-fold enhanced affinity at 4 "C (24), we measured the binding of UTP to the wild-type and mutant enzymes at this temperature? As seen in Fig. 5, even at 4 "C, the binding of UTP to the wild-type enzyme is just detectable by equilibrium dialysis; however, under identical conditions UTP binding to the Asp- 19r + Ala enzyme is enhanced 2-foldover the wild-type enzyme. UTP binds to approximately three sites on the Asp- 19r + Ala enzyme, although the number of binding sites cannot be accurately determined because of the low binding affinity of UTP. Binding of UTP in the Presence of CTP to the Mutant Enzyme Cannot Be Detected-The binding of UTP to the wild-type enzyme was measured in the presence of CTP. As mentioned above, in the absence of CTP, UTP binding to the wild-type enzyme cannot be detected at 25 "C by equilibrium dialysis suggesting that the dissociation constant of UTP is greater than 2 x However, at 25 "C in the presence of The synergistic inhibition of aspartate carbamoyltransferase by UTP in the presence of CTP is observed at 4 "C (data not shown).

Binding of UTPICTP to Aspartate Carbamoyltransferase 22157 CTP, the binding of UTP is enhanced, just as the binding of CTP is enhanced in the presence of UTP. Even under these conditions the binding of UTP is still weak. Although an accurate determination of the number of binding sites is not possible, the data are consistent with binding to three sites. In a parallel experiment, the binding of UTP to the Asp-19r -* Ala enzyme was measured in the presence of CTP. For the mutant enzyme, no UTP binding could be detected. ATP Binds to the Same Sites as UTP on the Wild-type and Mutant Enzymes-The inhibition of the wild-type enzyme by CTP alone or CTP plus UTP can be reversed by the addition of ATP (Fig. 6A). The enhanced affinity for the pyrimidine nucleotides when both CTP and UTP are present is obvious, for it requires much higher concentrations of ATP to reverse the inhibition. In a completely analogous experiment, the inhibition of the Asp-19r * Ala enzyme by CTP alone, and CTP plus UTP, can also be completely reversed by the addition of ATP (Fig. 6B). However, the reversal of the inhibition caused by ATP is almost identical whether or not UTP is present. These data indicate that UTP is not enhancing the affinity of the mutant enzyme for CTP. In order to probe the UTP inhibition of the Asp-19r 3 Ala enzyme in the absence of CTP, a similar competition experiment was performed. The Asp-19r + Ala enzyme was inhibited 20% by UTP (15 mm), and ATP was then varied in an attempt to displace UTP in a competitive manner. As seen in Fig. 6B, ATP can reverse the inhibition of the Asp-19r 3 Ala enzyme induced by UTP binding. The increased concentrations of ATP required to reverse the UTP inhibition is additional evidence for the enhanced binding of UTP (in the absence of CTP) to the mutant enzyme. Preliminary competition experiments by equilibrium dialysis also indicate that ATP directly causes loss of UTP binding (data not shown). DISCUSSION Previous work has shown that ATP and CTP compete for the same binding sites on the regulatory chains of wild-type aspartate carbamoyltransferase (22, 44, 45). The competition experiments (Fig. 6A) show that ATP competes for the UTP- [ATP], mm FIG. 6. Influence of ATP concentration on the activity of wild-type (A) and Asp-1% Ala (B) enzymes in the absence of nucleotides (0) and in the presence of UTP (A), CTP (0), and CTP plus UTP (A). Experimental conditions are the same as those reported in Fig. 2. For the competition experiment between UTP and ATP, the enzyme activity was measured in the presence of a concentration of UTP that gives 4 and 20% inhibition of the wildtype and the Asp-19r - Ala enzymes, respectively. For the other competition experiments sufficient nucleotide was added to give the maximum inhibition in each case, 2 mm for CTP alone, and 2 mm CTP plus 2 mm UTP. and CTP-binding sites suggesting that all the regulatory nucleotides bind to the same sites on the enzyme. The equilibrium dialysis experiments indicate that in the absence of UTP, CTP binds to all six of the nucleotide sites, three with high affinity and three with low affinity. However, the addition of UTP alters the binding substantially. In the presence of UTP, only three strong CTP sites are observed with significantly enhanced affinity. UTP binding experiments in the presence of CTP suggest approximately three binding sites with enhanced affinity over UTP binding in the absence of CTP. All of the experimental results reported here fit a model in which CTP occupies all the nucleotide sites on the regulatory chains of the wild-type enzyme in the absence of UTP, three with high affinity and three with low affinity. However, the binding of UTP, to presumably the low affinity CTP sites, enhances the binding of CTP to the high affinity sites. With both the low and high affinity sites occupied, the overall effect is enhanced binding of both nucleotides, although the mechanism by which this enhanced binding occurs is not clear. The functional effect of the binding of both CTP and UTP is the almost complete inactivation of the enzyme. The experimental data for the Asp-19r * Ala enzyme can easily be explained based on the above proposal if the mutation prevents or reduces the binding of UTP to the low affinity nucleotide sites and enhances the binding of UTP to the high affinity sites. Support for this comes from the equilibrium dialysis experiments that measure the binding of CTP in the absence and presence of UTP to the Asp-19r * Ala enzyme. The binding of CTP is nearly identical whether or not UTP is present and is identical to the binding of CTP to the wildtype enzyme in the absence of UTP (Fig. 5). The inhibition of the Asp-19r 4 Ala enzyme by UTP in the absence of CTP could simply be due to the ability of UTP to bind to the strong set of nucleotide sites on the mutant enzyme and inhibit the mutant enzyme in an analogous fashion as CTP inhibits the wild-type enzyme. The equilibrium dialysis experiments of the mutant enzyme support this hypothesis. The experiment shows enhanced affinity of UTP in the absence of CTP and no binding of UTP in the presence of CTP. However, these experiments cannot distinguish at which class of nucleotide sites binding is occurring. Based on the x-ray structure of the wild-type enzyme with ATP and CTP bound (31,32), there does not seem to be any side chains that couldbeused directly by the enzyme to distinguish between UTP and CTP. The overall geometry of the binding site must therefore be critical for discrimination between the nucleotides. In fact, the two classes of sites observed when CTP binds to the wild-type enzyme may be nature s mechanism for creating separate UTP and CTP sites. In addition to the side chain interaction between the carboxylate oxygen of Asp-19r and the ribose 3 OH there is also a salt link between Asp-19r and Lys-56r, a residue that has previously been shown to be important for normal regulatory function of the enzyme (46). When the salt link between Asp- 19r and Lys-56r is broken in the mutant enzyme, subtle conformational changes at the nucleotide site must result, thereby preventing the low affinity sites from binding UTP and allowing UTP to not only bind at the high affinity sites but also to inhibit the enzyme like CTP. X-ray crystallography of the Asp-19r + Ala enzyme is in progress to determine the molecular level details of this amino acid substitution in greater detail. Nevertheless, the use of site-specific mutagenesis in this case has dramatically extended our understanding of nucleotide binding to the wild-type enzyme and suggests that the low affinity CTP sites are the primary binding sites of UTP in wild-type aspartate carbamoyltransferase.

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