A Fluorescent Probe-labeled Escherichia coli Aspartate Transcarbamoylase That Monitors the Allosteric Conformational State*

A new system has been developed capable of monitoring conformational changes of the 240s loop of aspartate transcarbamoylase, which are tightly correlated with the quaternary structural transition, with high sensitiv-ity in solution. Pyrene, a fluorescent probe, was conjugated to residue 241 in the 240s loop of aspartate transcarbamoylase to monitor changes in conformation by fluorescence spectroscopy. Pyrene maleimide was conjugated to a cysteine residue on the 240s loop of a previously constructed double catalytic chain mutant version of the enzyme, C47A/A241C. The pyrene-labeled enzyme undergoes the normal T to R structural transition, as demonstrated by small-angle x-ray scattering. Like the wild-type enzyme, the pyrene-labeled enzyme exhibits cooperativity toward aspartate, and is acti-vated by ATP and inhibited by CTP at subsaturating concentrations of aspartate. The binding of the bisubstrate analogue N -(phosphonoacetyl)- L -aspartate (PALA), or the aspartate analogue succinate, in the presence of saturating carbamoyl phosphate, to the pyrene-labeled enzyme caused a sigmoidal change in the fluorescence emission. Saturation with ATP and CTP (in the presence of either subsaturating amounts of PALA or succinate nucleotide concentra- tion. The experimental data were analyzed in the same manner as the data from the activity assays. Small Angle X-ray Scattering— The SAXS experiments were performed at Beam Line 4-2 at the Stanford Synchrotron Radiation Lab- oratory (3.0 GeV, 50–100 mA). The experimental setup and procedures were performed as described (35, 36).

Allosteric regulation of enzymatic activity is manifested by the ability of the enzyme to exist in at least two different structural and functional forms (1). Enzymatic activity can therefore be modulated by changing the form in which the enzyme exists, by altering the dynamic equilibrium between multiple forms at a given time, or by causing more localized changes in the structure. Allosteric enzymes are also characterized by the regulation of their activity by effectors that bind at sites remote from the active sites.
Escherichia coli aspartate transcarbamoylase (EC 2.1.3.2) is a paradigm of allosteric enzymes in the study of allosteric regulation. This enzyme catalyzes the committed step of pyrimidine biosynthesis, the carbamoylation of the amino group of L-aspartate by carbamoyl phosphate to form N-carbamoyl-Laspartate and inorganic phosphate (2). Allosteric regulation is manifested in two different ways: homotropic cooperativity for the substrate L-aspartate and heterotropic regulation by ATP, CTP (2), and UTP in the presence of CTP (3). Aspartate transcarbamoylase from E. coli is a dodecamer composed of six catalytic (C) 1 chains organized as two trimeric subunits, and six regulatory (R) chains organized as three dimeric subunits. The active sites are shared between catalytic chains on the same trimer, and the nucleotide effectors bind to the same site on each of the regulatory chains (4 -8).
The two different structural and functional states of aspartate transcarbamoylase are the low affinity, low activity conformation of the enzyme, or the T state, and the high affinity, high activity conformation of the enzyme, or the R state (9 -12). Physical studies, including sedimentation velocity (9), x-ray crystallography (13,14), and small-angle x-ray solution scattering (SAXS) (15,16), have provided evidence of a quaternary structural change upon the binding of the natural substrates or substrate analogues. In the Thr to Arg quaternary structural transition, the enzyme elongates by at least 11 Å along the 3-fold axis, the upper and lower catalytic trimers rotate 15°r elative to one another, and the regulatory dimers rotate 15°a round their respective 2-fold axes (17,18). In addition to these 1 The abbreviations used are: C, catalytic chain; R, regulatory chain; SAXS, small-angle X-ray scattering; PALA, N-(phosphonoacetyl)-L-aspartate; [Asp] 0.5 , the aspartate concentration at half the maximal observed specific activity; C47A/A241C, the double mutant aspartate transcarbamoylase holoenzyme with Cys-47 replaced by Ala and Ala-241 replaced by Cys in each of the catalytic chains; C47A/A241Cpyrene, the double mutant labeled with pyrene; C47A/A241C-c, the double mutant aspartate transcarbamoylase catalytic subunit with Cys-47 replaced by Ala and Ala-241 replaced by Cys in each of the catalytic chains; C47A/A241C-c-pyrene, the double mutant catalytic subunit labeled with pyrene; 240s loop, a loop in the catalytic chain of aspartate transcarbamoylase comprised of residues 232-246; F c , fluorescence coefficient. quaternary structural changes, several tertiary changes also occur during the T to R state transition. One example of a tertiary change that is of importance to this study is the reorientation of the 240s loop, which facilitates the domain closure in the catalytic chains, resulting in the formation of high activity high affinity active sites (19). In particular, the position of the two 240s loops at the C1-C4 2 interface changes from lying sideways and apart in the T state, to a position in which the loops are stacked on top of one another in the R state (see Fig. 1). Thus, the quaternary structural transition is tightly correlated with the conformational change of the 240s loop.
To create a fluorescent-labeled enzyme, which could monitor the allosteric transition, we conjugated a fluorophore site specifically via a Cys residue at position 241 of the 240s loop ( Fig.  1). Previous studies have shown that when Ala-241 in the wild-type enzyme is converted to Cys the modified enzyme has wild-type like properties (20). The fluorophore used in this study, pyrene, fluoresces not only from an excited monomer state, but also from an excited-state dimer, or excimer; excimer fluorescence results from a specific interaction between the excited monomer and a ground-state monomer (21). Pyrene site specifically attached to multiple sites on a protein has been utilized in a number of studies as a "molecular ruler" with a range of ϳ0 -20 Å. Hence it is capable of sensitively monitoring a conformational change in proteins (22,23). The 240s loop was chosen as the site of attachment for pyrene because of the relatively large conformational change it undergoes in the T to R state transition (13,24). As shown in Fig. 1, the excimer should only form in the R state, thus providing a unique and specific signal for the R state. Here we used the pyrene-labeled aspartate transcarbamoylase to monitor the allosteric transition and the movement of the 240s loop into the R state conformation, induced by the binding of substrates, substrate analogues, and allosteric effectors.
Overexpression and Purification of the Mutant Enzymes-The C47A/ A241C mutant and wild-type holoenzymes, and C47A/A241C catalytic subunit of aspartate transcarbamoylase were overexpressed and purified to homogeneity as previously described (25). After concentration, the purity of the enzymes was checked by non-reducing (in which dithiothreitol is omitted from the sample buffer) SDS-PAGE (26) and nondenaturing PAGE (27,28).
Protein Concentration-The concentration of the wild-type holoenzyme was determined by A 280 nm based upon an extinction coefficient of 0.59 cm 2 mg Ϫ1 (29). The concentration of the C47A/A241C-pyrenelabeled enzyme was determined by the Bio-Rad version of the Bradford dye binding assay (30).
Fluorescent Labeling of the C47A/A241C Holoenzyme and C47A/ A241C-c Catalytic Subunit-Fluorescent labeling of the mutant enzymes was performed according to the procedure provided by Molecular Probes. The purified mutant enzymes were concentrated to ϳ20 mg/ml and dialyzed overnight into 40 mM KH 2 PO 4 buffer, pH 7.2. After dialysis, a 100-fold molar excess of solid tris-(2-carboxyethyl)phosphine hydrochloride was added with stirring to the enzyme solution, at 20°C. One hour after addition of the tris-(2-carboxyethyl)phosphine hydrochloride, a freshly prepared 20 mM solution of N-(1-pyrene)maleimide in N,N-dimethylformamide in 60-fold molar excess was slowly dripped into the vigorously stirred enzyme solution. After addition of the N-(1pyrene)maleimide solution, the reaction mixture was shielded from light by covering the reaction vessel with foil. After 2 h, the reaction mixture was centrifuged to pellet out the N-(1-pyrene)maleimide. The supernatant was dialyzed four times against 40 mM KH 2 PO 4 buffer, pH 7.0, 2 mM dithiothreitol, and 1 mM EDTA to remove the unreacted N-(1-pyrene)maleimide.
Efficiency of Pyrene Attachment-The completeness of the labeling of the C47A/A241C enzyme with pyrene was assessed by mass spectrometry and also by determination of unreacted thiol with Ellman's reagent (31). The C47A/A241C and C47A/A241C-pyrene holoenzymes were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry using a Micromass TofSpec-2E mass spectrometer operating in positive mode and using a sinaptic acid matrix.
Aspartate Transcarbamoylase Assay-The aspartate transcarbamoylase activity was measured at 25°C by the colorimetric method (32). Saturation curves were performed in duplicate, and data points shown in the figures are the average values. Assays were performed in 50 mM Tris acetate buffer, pH 8.3, in the presence of saturating carbamoyl phosphate (4.8 mM). Data analysis of the steady-state kinetics was carried out as described previously (33). Fitting of the experimental data to theoretical equations was accomplished by non-linear regression. When substrate inhibition was negligible, data were fit to the Hill equation. If substrate inhibition was significant, data were analyzed using an extension of the Hill equation that included a term for substrate inhibition (34). The nucleotide saturation curves were fit to a hyperbolic binding isotherm by non-linear regression.
Fluorescence Measurements-Fluorescence emission spectra were recorded at 20 Ϯ 1°C in 50 mM Tris acetate buffer, pH 8.3, on a Shimadzu 1000 spectrofluorimeter. The excitation wavelength was 338 nm; the emission wavelength collected was 360 -560 nm; the excitation and emission bandwidths were set to 5 nm. To approximate the degree of quaternary structure change for the enzyme population from the fluorescence emission spectra, a unitless fluorescence coefficient (F c ) was calculated from the following expression,

Fluorescent Pyrene-labeled Aspartate Transcarbamoylase
where I F is the fluorescence intensity at wavelength i sampled over the entire emission spectrum at 0.4-nm intervals. The ratio of the sums of the intensities of the excimer to the monomer fluorescence was used to significantly reduce the signal to noise. All fluorescence experiments were performed with the enzyme in 50 mM Tris acetate buffer, pH 8.3.
Saturation curves with the bisubstrate analogue PALA and aspartate analogue succinate (plus saturating carbamoyl phosphate (4.8 mM)) were performed by incremental addition to the pyrene-labeled enzyme ([C47A/A241C-pyrene] ϭ 1 M) until there was no further change in the fluorescence emission spectrum. To normalize the data, the F c for all data points was divided by the F c at 0 ligand (PALA or succinate) concentration, and then subtracted by 1. The experimental data were analyzed in the same manner as the data from the activity assays.
Nucleotide saturation curves ([C47A/A241C-pyrene] ϭ 1 M) were performed at a subsaturating PALA concentration ([PALA]:[C47A/ A241C-pyrene] ϭ 1), a subsaturating succinate concentration ([succinate] ϭ 1 mM and [carbamoyl phosphate] ϭ 4.8 mM), and also in the absence of ligands. ATP or CTP were added incrementally until the enzyme was completely saturated with nucleotide, as indicated by no further change in the fluorescence spectrum. To normalize the data, the F c for all data points was divided by the F c at 0 nucleotide concentration. The experimental data were analyzed in the same manner as the data from the activity assays.
Small Angle X-ray Scattering-The SAXS experiments were performed at Beam Line 4-2 at the Stanford Synchrotron Radiation Laboratory (3.0 GeV, 50 -100 mA). The experimental setup and procedures were performed as described (35,36).

A Cys Residue on the 240s Loop for Covalent Attachment of a Fluorescent Probe-
The distance between identical residues in the 240s loop in the C1 and C4 chains of aspartate transcarbamoylase changes significantly in the transition from the T to R states (13,24). Therefore, the 240s loop was chosen as the location for placement of a fluorescent probe that is sensitive to conformation. We chose to conjugate pyrene site specifically to our previously constructed 240s loop mutant, C47A/A241C (20). The substitution of the one naturally occurring cysteine in the catalytic chain, Cys-47, by alanine ensured site-specific labeling.
After labeling the C47A/A241C holoenzyme with pyrene, the degree of labeling as determined by mass spectrometry appeared nearly complete. The molecular mass of the catalytic chain for the C47A/A241C holoenzyme, as determined by mass spectrometry, was 33,958 Da. The mass spectrum for the C47A/ A241C-pyrene holoenzyme showed a single peak for the catalytic chain, which had a molecular mass of 34,292 Da. This is a difference in molecular mass of 342 Da, close to the molecular mass of pyrene maleimide (297.3). The degree of labeling with pyrene, as determined by the amount of unreacted thiol using Ellman's reagent, was 70 Ϯ 10%. The fluorescence emission spectrum of the C47A/A241C-pyrene-labeled holoenzyme is shown in Fig. 2A.
Steady-state Kinetics of the Wild-type and Fluorophore-labeled Enzymes-The kinetic parameters calculated from the aspartate saturation curves shown in Fig. 3A are displayed in Table I. The parameters for wild-type holoenzyme were a maximal activity of 17.5 mmol h Ϫ1 mg Ϫ1 , an [Asp] 0.5 of 13.0 mM, and Hill coefficient of 2.4. The C47A/A241C-pyrene-labeled holoenzyme exhibited a sigmoidal aspartate saturation curve similar to that of the wild-type curve, with a maximal activity of 15.8 mmol h Ϫ1 mg Ϫ1 , an [Asp] 0.5 of 12.5 mM, and somewhat reduced cooperativity with a Hill coefficient of 1.4.

Influence of the Allosteric Effectors on the Wild-type and
Labeled Enzymes-Nucleotide saturation curves with either CTP or ATP were determined for the wild-type and C47A/ A241C-pyrene-labeled holoenzymes at one-half the [Asp] 0.5 , because the effect of the nucleotides are observed only at subsaturating concentrations of aspartate. This concentration of aspartate was selected because the nucleotides exert a larger influence on the activity of the enzyme as the aspartate concentration is reduced (37). Based upon the nucleotide saturation curves shown in Fig. 4, the maximal extent of activation or inhibition at infinite nucleotide concentration was calculated. The parameters obtained from fitting these curves are given in Table II. ATP activates and CTP inhibits the C47A/A241Cpyrene-labeled holoenzyme. However, the magnitude of the nucleotide effect is reduced both for ATP and CTP to approximately one-half that exhibited by the wild-type holoenzyme. Considering that the same reduction in the heterotropic effects of ATP and CTP was observed for the unlabeled C47A/A241C holoenzyme (20), it can be concluded that the reduced effects are because of the mutations and not to the pyrene labeling.
Monitoring the Structural Change by Steady-state Fluorescence-To determine whether the fluorescence of the C47A/ A241C-pyrene-labeled holoenzyme was sensitive to alterations in quaternary structure, steady-state fluorescence emission spectra were recorded in the absence and presence of PALA. There was a significant change in the fluorescence emission peaks with increasing PALA concentrations (see Fig. 2A). The intensity of the peak at 380 nm decreased, concomitant with the increasing intensity of a broad peak at ϳ480 nm. Shown in Fig. 2B is a plot of the F c of the C47A/A241C-pyrene-labeled holoenzyme as a function of PALA concentration. The sigmoidal data were fit to the Hill equation that gave a Hill coefficient The change in fluorescence emission of the C47A/A241Cpyrene-labeled holoenzyme was measured as a function of succinate concentration in the presence of saturating carbamoyl phosphate (see Fig. 3B). The change in the F c with increasing succinate concentration was also sigmoidal with a Hill coefficient of 2.0.
The change in fluorescence emission of the C47A/A241Cpyrene-labeled holoenzyme was also measured as a function of the concentration of the nucleotide effectors ATP and CTP. Saturating with ATP caused little change in the fluorescence emission, as the F c increased only ϳ2%. Saturating with CTP also caused no change in the fluorescence emission. To observe the influence of the nucleotides on any structural alterations, experiments were also performed at a subsaturating concen-tration of PALA or succinate (plus saturating carbamoyl phosphate), setting the [T]/[R] ratio close to 1. With either PALA or succinate, the F c increased with increasing ATP concentration, and decreased with increasing CTP concentration. When the fluorescence curve ([PALA]:[C47A/A241C-pyrene] ϭ 1) from the ATP saturation was normalized to the ATP saturation curve (kinetic assay), the curves could be overlaid almost exactly (see Fig. 4). From the ATP saturation fluorescence curves for both succinate and PALA, the half-saturation values (K ATP ϭ 1.3 Ϯ 0.6) were equal, but the F c values at saturating [ATP] was greater with succinate (85 versus 50%) than with PALA. When the fluorescence curve ([PALA]:[C47A/A241C-pyrene] ϭ 1) from the CTP saturation was normalized to the CTP saturation curve (kinetic assay), the curves also could be overlaid almost exactly (see Fig. 4). From the CTP saturation fluorescence curves for both succinate and PALA, the half-saturation values (K CTP ϭ 0.029 Ϯ 0.002) and the F c values at a saturating CTP concentration were equal (81%).
Small-angle X-ray Scattering-SAXS was used to evaluate the quaternary structures of the wild-type holoenzyme and the  The data for the C47A/A241C is reported under reducing conditions that prevent the cysteines from forming a disulfide bond between corresponding residues in the upper and lower catalytic subunits (20).  C47A/A241C-pyrene holoenzyme in the absence and presence of saturating concentrations of PALA (see Fig. 5). The data for the wild-type holoenzyme, shown in Fig. 5A, displays the characteristic change in the scattering pattern upon addition of PALA, as noted by the change in the peak position and increase in relative intensity (15) accompanying the T to R state transition. The scattering patterns of the C47A/A241C-pyrene holoenzyme are shown in Fig. 5B. The unliganded C47A/A241Cpyrene-labeled holoenzyme displayed the same characteristic change in the peak position and relative intensity of the scattering pattern as the wild-type holoenzyme from addition of PALA.
Monitoring the Structural Change by Stopped-flow Fluorescence and SAXS-The time evolution of the change in fluorescence induced by the binding of the natural substrates carbamoyl phosphate and aspartate to the C47A/A241C-pyrene-labeled holoenzyme was monitored by stopped-flow fluorescence. As seen in Fig. 6, when a saturating concentration of aspartate was mixed with the C47A/A241C-pyrene-labeled holoenzyme saturated with carbamoyl phosphate the fluorescence changes and then levels off. To correlate the change in fluorescence with the quaternary structural change, stopped-flow SAXS was employed (36). The relative change in the SAXS intensity, when a saturating concentration of aspartate was mixed with C47A/ A241C-pyrene-labeled holoenzyme saturated with carbamoyl phosphate, was very similar to that observed by fluorescence. The buffer and pH used for the florescence and stopped-flow SAXS were identical; however, the SAXS experiment required an enzyme concentration ϳ500-fold (35 mg/ml) higher than used for the fluorescence experiments. DISCUSSION Aspartate transcarbamoylase has two allosteric states, T and R, each distinct in structure and function. The quaternary structural transition from the low affinity, low activity T state to the high affinity, high activity R state upon binding substrate analogues has been well demonstrated (9,14,24,38,39). However, details of the structural transition between the T and R states have been difficult to obtain. To be able to observe the actual T to R transition in aspartate transcarbamoylase, as well as to monitor structural changes induced by the heterotropic effectors, we have developed a fluorescent-labeled version of aspartate transcarbamoylase that not only has kinetic characteristics similar to the wild-type enzyme, but also fluoresces uniquely in the R quaternary structure.
The fluorescent label was attached to the enzyme site-specifically in the 240s loop of the catalytic chain of the enzyme because the 240s loops at the C1:C4 interface are much closer in the R state than in the T state (13,24). For instance, the distance between side chains for a pair of identical residues in the region 236 -241 at the C1:C4 interface is greater than 20 Å in the T state, whereas this distance becomes less than 10 Å in the R state. Pyrene was selected as the fluorophore because it absorbs in the near UV region, and is sensitive to the proximity of another pyrene, as a large change in the fluorescence emission spectrum occurs upon excimer formation (21). The fluorescence emission spectrum of pyrene is characterized by a monomeric emission peak at 380 nm, which decreases with a concomitant increase in a broad peak at 480 nm upon excimer formation. This excimer emission occurs only when two pyrenes are within ϳ10 Å of one another (21). Previous fluorescence studies of aspartate transcarbamoylase were performed in which the Tyr-240 was substituted by Trp in the 240s loop; and this mutant enzyme was sensitive to the conformational change of the 240s loop (40,41), but the fluorescence changes were not directly correlated with the quaternary structural transition as determined by SAXS.
We chose to substitute a Cys residue into the 240s loop for covalent attachment of pyrene. However, because many of the residues in the 240s loop are critical for the proper allosteric regulation of this enzyme, such as Asp-236 (42) and Glu-239 (43), and because we sought a system that closely resembled the kinetics of the wild-type enzyme, our choices for residues to mutate to Cys were limited. Therefore, we chose as the best candidate a double mutant enzyme previously created in our laboratory, C47A/A241C, which was known to have kinetic parameters very similar to that of the wild-type enzyme (20). Site-specific labeling with pyrene at Cys-241 was assured as the single cysteine in the wild-type catalytic chain, Cys-47, had been replaced with Ala. The mass spectrometry and the determination of unreacted thiol with Ellman's reagent suggest that most of the C47A/A241C-pyrene enzyme was labeled.
The C47A/A241C-pyrene enzyme was tested in several experiments to determine whether it behaved as does the wildtype enzyme. The aspartate saturation curve of C47A/A241Cpyrene was sigmoidal, and the maximal velocity and [Asp] 0.5 were close to the wild-type values ( Table I). The nucleotide saturation curves of C47A/A241C-pyrene demonstrated activation by ATP and inhibition by CTP in a manner similar to that of the wild-type enzyme, albeit with lower activation by ATP and lower inhibition by CTP (Table II). The unliganded C47A/ A241C-pyrene enzyme (Fig. 5B) exhibited a SAXS pattern similar to that of the wild-type enzyme in the absence of ligands (Fig. 5A). In the presence of PALA, the C47A/A241C-pyrene enzyme demonstrated the same shift in peak position and relative intensity as did the wild-type enzyme in the presence of PALA indicating that the enzyme undergoes the allosteric transition.
Because the pyrenes on the 240s loops should be much closer in the R state than the T state, as shown in our model of the C1 and C4 catalytic chains conjugated with pyrene at Cys-241 in Fig. 1, excimer formation should only occur in the R state. Excimer formation caused by the T to R state transition upon addition of PALA was successfully demonstrated (see Fig. 2A). In addition, as shown in Figs. 2B and 3B, the increase in the F c upon saturation with PALA or succinate (in the presence of saturating carbamoyl phosphate) is sigmoidal, demonstrating the cooperative nature of PALA and succinate binding, analogous to the cooperative binding of the substrate aspartate. The fluorescence change is complete only after six molecules of PALA per molecule of holoenzyme have been added, as opposed to the structural change as monitored by SAXS that is complete by the addition of ϳ3 PALA molecules per holoenzyme. These results indicate that the allosteric transition of aspartate transcarbamoylase is composed of linked global and local changes in structure. For fluorescence to be observed not only does the enzyme have to be in the expanded quaternary structure, but also local changes in the 240s loop must occur. The conformational changes associated with the binding of PALA have been observed by x-ray crystallography in both in the holoenzyme (44) as well as the isolated catalytic subunit (45).
To confirm that the change in fluorescence emission observed upon addition of PALA to the C47A/A241C-pyrene holoenzyme was caused by the quaternary structural change and closure of the 240s loops after the binding of the bisubstrate analogue PALA, and not simply the binding of PALA to the active site, the isolated C47A/A241C-c catalytic subunit was also labeled with the pyrene fluorophore. No excimer formation was observed when the C47A/A241C-c-pyrene catalytic subunit was saturated with PALA. In the case of the isolated catalytic subunit the closure of the domains of the catalytic chains is a requirement for the formation of the active site and catalysis. However, the conformational changes that occur in the isolated catalytic subunit cannot occur in the holoenzyme without an expansion of the enzyme along the 3-fold axis, because the closure of the domains and the movement of the 240s loops is impossible because of steric constraints imposed by a catalytic chain in the opposite catalytic subunit.
To correlate the quaternary structural change with the change in fluorescence because of the formation of the excimer in the presence of the natural substrates, stopped-flow SAXS was employed. As seen in Fig. 6, there is a close correlation between the rate of the structural change observed by stopped-flow SAXS and the rate of change in the fluorescence change. Because saturating concentrations of substrates were used in these experiments all of the enzyme active sites should be filled, as opposed to the data in Fig. 2 in which the observed fluorescence was monitored as a function of substrate saturating. These data support the conclusion that excimer formation directly reflects the alterations in quaternary structure of the enzyme.
Because of the ordered binding of the substrates in aspartate transcarbamoylase, the allosteric transition is monitored as a function of aspartate concentration at a saturating concentration of carbamoyl phosphate. The binding of aspartate requires the closure of the two domains of the catalytic chain and the repositioning of the 80s and 240s loops resulting in the high activity, high affinity active site. However, this domain closure cannot take place in a single active site because the movement of the domains in an upper catalytic chain is blocked by the corresponding domains in a lower catalytic chain (44). Thus, the binding of aspartate in the presence of carbamoyl phosphate induces the allosteric transition (44). The results reported here as well as Trp fluorescence experiments (40) indicate that once the quaternary structural change has occurred, the 240s loop positions in the catalytic chains that have not bound aspartate must be a different conformation from the ones that have bound aspartate. Thus, the allosteric transition in aspartate transcarbamoylase is composed of the quaternary structural change accompanied by local conformational changes near the active sites. The local conformational changes are not induced by the quaternary structural change but rather occur on a site by site basis induced by the binding of substrates to each of the high affinity active sites created by the quaternary structural change. The change in the fluorescence observed upon addition of PALA to the C47A/A241C-pyrene-labeled holoenzyme suggests that the global structural change puts the enzyme into the R quaternary structure and primes the active sites for substrate binding. However, it is the binding of PALA, or aspartate in the presence of carbamoyl phosphate, that causes the final repositioning of the 240s loop into its catalytically active conformation. These local conformational changes may actually occur in a transient fashion to allow product release after the reaction has taken place.
To examine whether the effect exerted by the nucleotides would cause a change in the fluorescence emission, fluorescence spectra were recorded for the C47A/A241C-pyrene holoenzyme at a saturating concentration of ATP or CTP. ATP induced only a very small increase in the F c , whereas CTP induced no measurable decrease in the F c . This agrees with structural studies that show that ATP and CTP do not appreciably change the quaternary structure of the enzyme (46). The influence of ATP and CTP was also examined after addition of a subsaturating amount of PALA or succinate, as was done in SAXS experiments that measured the effect of the nucleotides on the wild-type enzyme (47,48). In the first study, the authors observed an effect on the [T]/[R] equilibrium for both ATP and CTP when succinate and carbamoyl phosphate were used. However, they detected no effect by ATP on the [T]/[R] equilibrium when PALA was used. This equilibrium shift in the case of ATP was interpreted as a secondary effect caused by a change in affinity for the substrate analogue, succinate. As demonstrated by our fluorescence data, shown in Fig. 4, the F c increased in a hyperbolic manner upon saturation with ATP in the presence of a subsaturating concentration PALA. In addition, when the fluorescence curve from the ATP saturation was normalized to the ATP saturation curve (kinetic assay), the curves could be overlaid almost exactly. There was a greater effect upon the fluorescence emission by ATP with succinate than with PALA, with an increase in the F c of 85%. In addition, when the fluorescence curve from the CTP saturation was normalized to the CTP saturation curve (kinetic assay), the curves overlaid exactly (Fig. 4). The effect of CTP upon the fluorescence emission was the same with succinate and PALA.
Because of the strong evidence, from the SAXS experiments (47,48) that the nucleotide ATP does not perturb the [T]/[R] equilibrium, our results suggest that the effect of ATP on the activity of aspartate transcarbamoylase is to cause a local change in the conformation of the 240s loop in the R state. We suggest that the conformational change is the closure of the 240s loops in the catalytic chains that do not have substrate bound to form the high affinity, high activity active site. The effect of CTP upon activity appears to be caused by a perturbation of the [T]/[R] equilibrium, which both our fluorescence results and the SAXS experiments suggest. Another possibility concerning the effect of CTP is that in addition to the structural equilibrium shift the opposite effect of ATP on the conformation of the 240s loops occurs, causing an opening of the 240s loops in the R state to decrease the number of catalytic chains with the fully formed active site. Thus, the fluorescence changes observed by the addition of ATP and CTP are directly related to the position of the 240s loop as it either helps to create the active site (ATP) or prevents the active site from forming (CTP).
The availability of the pyrene-labeled enzyme developed here provides a new tool for the study of aspartate transcarbamoylase and the structural changes that the molecule undergoes. For example, the quaternary conformation of the enzyme that is trapped in silica matrix sol-gels can be directly determined (49) and fluorescence stopped-flow will not only allow the direct monitoring of the allosteric conformational change but also allow a means to directly determine how the regulatory nucleotides alter the rate of the allosteric transition.