The Role of Intersubunit Interactions for the Stabilization of the T State of Escherichia coli Aspartate Transcarbamoylase*

Homotropic cooperativity in Escherichia coli aspartate transcarbamoylase results from the substrate-induced transition from the T to the R state. These two alternate states are stabilized by a series of interdomain and intersubunit interactions. The salt link between Lys-143 of the regulatory chain and Asp-236 of the catalytic chain is only observed in the T state. When Asp-236 is replaced by alanine the resulting enzyme exhibits full activity, enhanced affinity for aspartate, no cooperativity, and no heterotropic interactions. These characteristics are consistent with an enzyme locked in the functional R state. Using small angle x-ray scattering, the structural consequences of the D236A mutant were characterized. The unliganded D236A holoenzyme appears to be in a new structural state that is neither T, R, nor a mixture of T and R states. The structure of the native D236A holoenzyme is similar to that previously reported for another mutant holoenzyme (E239Q) that also lacks intersubunit interactions. A hybrid version of aspartate transcarbamoylase in which one catalytic subunit was wild-type and the other had the D236A mutation was also investigated. The hybrid holoenzyme, with three of the six possible interactions involving Asp-236, exhibited homotropic cooperativity, and heterotropic interactions consistent with an enzyme with both T and R functional states. Small angle x-ray scattering analysis of the unligated hybrid indicated that the enzyme was in a new structural state more similar to the T than to the R state of the wild-type enzyme. These data suggest that three of the six intersubunit interactions involving D236A are sufficient to stabilize a T-like state of the enzyme and allow for an allosteric transition.

dodecamer composed of six catalytic chains of M r 34,000 and six regulatory chains of M r 17,000. The catalytic chains are organized as two trimeric subunits (C), 1 whereas the regulatory chains are organized as three dimeric subunits (R). The active sites are located at the interfaces between adjacent catalytic chains, whereas the nucleotide effectors bind to the same site on each of the regulatory chains (4 -8).
Two functionally and structurally different states of aspartate transcarbamoylase have been characterized. The low affinity, low activity conformation of the enzyme is described as the T state and the high affinity, high activity conformation of the enzyme is described as the R state. The conversion from the T to the R state occurs upon aspartate binding to the enzyme in the presence of carbamoyl phosphate. Structurally, the enzyme elongates by at least 11 Å along the 3-fold axis, the upper catalytic trimer rotates 10°relative to the lower trimer, and the regulatory dimers rotate 15°around the 2-fold axes (9,10). In addition to these quaternary changes, several tertiary changes also occur during the T to R state transition. In particular, the 80s and 240s loops reorient. The distinct interchain contacts of side chains of the 240s loop in the T and R states have been identified as being the major contributors to the stabilization of the T and R states (11).
The manifestation of homotropic cooperativity in aspartate transcarbamoylase results from the substrate-induced transition from the T state to the R state. These two alternate structural and functional states are stabilized by a series of interdomain and intersubunit interactions. For example, the two domains of the catalytic chain are stabilized in their domain-closed R conformation by stabilizing interactions between Glu-50 of the carbamoyl phosphate domain to Arg-167 and Arg-229 of the aspartate domain (Fig. 1). These interdomain bridging interactions are critical for the formation and stabili- 1 The abbreviations used are: C, catalytic subunit of aspartate transcarbamoylase composed of three catalytic chains; R, regulatory subunit of aspartate transcarbamoylase composed of two regulatory chains; PALA, N-(phosphonoacetyl)-L-aspartate; [Asp] 0.5 , the aspartate concentration at half-maximal observed specific activity; AT-C, the mutant catalytic subunit in which 6 aspartate residues have been added to the C terminus of each catalytic chain; (AT-C) 2 R 3 , the reconstituted mutant holoenzyme in which 6 aspartate residues have been added to the C terminus of each catalytic chain; (D236A-C) 2 R 3 , reconstituted mutant holoenzyme with Ala substituted in place of Asp-236 in each catalytic chain; (D236A-C)(AT-C)R 3 , mutant hybrid holoenzyme in which one catalytic subunit has Ala substituted in place of Asp-236 in each catalytic chain and the other catalytic subunit has 6 aspartate residues added to the C terminus of each catalytic chain; native D236A holoenzyme, the aspartate transcarbamoylase holoenzyme in which each of the Asp-236 residues in the catalytic chains has been replaced by alanine (this enzyme was not produced by reconstitution and is equivalent to that previously reported (1)); SAXS, small angle X-ray scattering. A similar set of abbreviations is used for the holoenzyme and hybrid with the E239Q mutation. zation of the R state of the enzyme (11). Interactions between catalytic chains in different catalytic subunits play an important role in T state stabilization. When the links between Glu-239c1 2 and both Lys-164c4 and Tyr-165c4 are disrupted, the T state of the enzyme is no longer stable, resulting in the formation of a stable intermediate structural state (12). Links between the catalytic and regulatory subunits are also important for T state structural stabilization (Fig. 1). For example, breaking of the link between Lys-143r1 and Asp-236c4 results in an enzyme with full activity, enhanced affinity for aspartate, no cooperativity, no activation by ATP, and minimal inhibition by CTP (13,14). These functional characteristics are consistent with an enzyme locked in the functional R state. Here we characterize structurally the D236A holoenzyme and characterize both functionally and structurally a hybrid version of aspartate transcarbamoylase that has only three of the six possible intersubunit interactions between Lys-143r and Asp-236c, to more fully understand the functional role of the interactions between the catalytic and regulatory chains of aspartate transcarbamoylase.

EXPERIMENTAL PROCEDURES
Materials-Agarose, ATP, CTP, L-aspartate, N-carbamoyl-L-aspartate, potassium dihydrogen phosphate, and uracil were obtained from Sigma. Ampicillin, Tris, Q-Sepharose fast flow resin, and Source Q resin were purchased from Amersham Biosciences. UNO Q-1, protein assay dye, and sodium dodecyl sulfate were purchased from Bio-Rad. Carbamoyl phosphate dilithium salt, obtained from Sigma, was purified before use by precipitation from 50% (v/v) ethanol and was stored desiccated at Ϫ20°C (2). Casamino acids, yeast extract, and tryptone were obtained from Difco. Ammonium sulfate and electrophoresis grade acrylamide were purchased from ICN Biomedicals. Antipyrine was obtained from Fisher.
Overexpression and Purification of Aspartate Transcarbamoylase Regulatory Subunit-The aspartate transcarbamoylase regulatory subunit was overexpressed utilizing strain EK1104 containing plasmid pEK168 (16). Bacteria were cultured at 37°C with agitation in M9 media containing 0.5% casamino acids, 12 g/ml uracil, and 150 g/ml ampicillin. Cells were harvested and resuspended in 0.1 M Tris-Cl buffer, pH 9.2, 0.1 mM zinc chloride followed by sonication to lyse the cells. The purification of the regulatory subunit was performed as previously described (16). Formation and Purification of Reconstituted Mutant Holoenzymes-Equal amounts of purified D236A-C and AT-C subunits were mixed with excess regulatory subunit and dialyzed overnight against 50 mM Tris acetate buffer, pH 8.3, 2 mM 2-mercaptoethanol, and 0.1 mM zinc acetate (22). The mixture was examined by nondenaturing PAGE to confirm the formation of the three holoenzyme species, (D236A-C) 2 R 3 , (D236A-C)(AT-C)R 3 , and (AT-C) 2 R 3 . After being verified by nondenaturing PAGE, the hybrid mixture was dialyzed into phosphate buffer (40 mM KH 2 PO 4 , pH 7.0) at 4°C in a preparation for separation using a Source Q anion exchange column (1 ϫ 8.2 cm). The enzyme was eluted off the column using a linear salt gradient (40 ml total volume) in phosphate buffer, 0 to 0.5 M NaCl over 40 min. The fractions were analyzed by using nondenaturing PAGE. The fractions containing the hybrid were pooled and concentrated. This pool was redialyzed into phosphate buffer and repurified a second time on the same Source Q column to remove trace impurities of the other reconstituted species.
Determination of Protein Concentration-The concentration of the wild-type holoenzyme, catalytic subunit, and regulatory subunit were determined from absorbance measurements at 280 nm using extinction coefficients of 0.59, 0.72, and 0.32 cm 2 mg Ϫ1 , respectively (23). The concentrations of the mutant enzymes were determined by the Bio-Rad version of the Bradford dye binding assay (24).
Determination of Nucleotide Concentration-The concentrations of the nucleotides was determined from absorbance measurements at the max at pH 7.0 using the respective molar extinction coefficients of the nucleotides.
Aspartate Transcarbamoylase Assay-The aspartate transcarbamoylase activity was measured at 25°C by the colorimetric method (25). 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. Data analysis of the steady-state kinetics was carried out as previously described (26). Fitting of the experimental data to theoretical equations was accomplished by nonlinear 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 (27). The nucleotide saturation curves were fit to a hyperbolic binding isotherm by nonlinear regression.
Small angle X-ray Scattering-The small angle x-ray scattering experiments were performed on the Beamline 4-2 at the Stanford Synchrotron Radiation Laboratory (3.0 GeV, 50 -100 mA). A significantly upgraded version of the small angle scattering instrument was used. The specimen to detector distance was 95 cm, and the x-ray wavelength was tuned to 1.381 Å using a Si (111) double-crystal monochromator (28). A linear gas chamber detector filled with a Xe/CO 2 mixture was used in the experiment. Total counting rate on the detector was between 30,000 and 90,000 counts/s. The scattering curves are expressed as the momentum transfer h (h ϭ 4(sin)/), where 2 and are the scattering angle and the wavelength of the x-ray beam, respectively), which was calibrated using the (100) reflection from a cholesterol myristate powder sample held at the specimen position. Sample solutions were maintained at 25°C. All scattering curves were normalized to incident beam intensity integrated over exposure time, and the corresponding solvent scattering was subtracted. The enzyme solution was adjusted so all the scattering curves were performed at an identical protein concentration.

RESULTS
Formation, Purification, and Steady-state Kinetics of (D236A-C) 2 R 3 and (D236A-C)(AT-C)R 3 -To produce an aspartate transcarbamoylase with only three of the six Lys-143r-Asp-236c interactions, a hybrid was constructed that had one wild-type catalytic subunit and one catalytic subunit with Asp-236 replaced by alanine (D236A-C). The (D236A-C)(AT-C)R 3 hybrid was formed by reconstitution of the holoenzyme from D236A-C, modified wild-type catalytic subunits (AT-C), and excess regulatory subunits (R). The isolation of the (D236A-C)(AT-C)R 3 hybrid is made possible because the modified wildtype catalytic subunits have a six-aspartic acid extension on the C terminus of each of the catalytic chains that serves as a chromatrographic handle. We have previously shown that the addition of these aspartic acid residues does not alter the kinetics of the catalytic trimer (AT-C) or the holoenzyme formed upon reconstitution (AT-C) 2 R 3 (16). Hybrid formation was verified by nondenaturing PAGE and the three resulting species were purified using anion-exchange chromatography (see Fig. 2).
Kinetic characterization of the (D236A-C)(AT-C)R 3 and (D236A-C) 2 R 3 holoenzymes were performed (Fig. 3). As shown in Table I, the reconstituted (D236A-C) 2 R 3 holoenzyme exhibits kinetics similar to the native D236A holoenzyme (13). The aspartate saturation curve of the (D236A-C) 2 R 3 holoenzyme exhibits no cooperativity with a maximal velocity of 20.3 mmol h Ϫ1 mg Ϫ1 and a K m of 1.1 mM. Under these conditions, substrate inhibition was observed similar to the wild-type enzyme. The aspartate saturation curve of the (D236A-C)(AT-C)R 3 hybrid holoenzyme exhibited slight cooperativity (n H ϭ 1.2) with a maximal velocity of 21.6 mmol h Ϫ1 mg Ϫ1 and an [Asp] 0.5 of 3.2 mM.
Influence of the Allosteric Effectors-Nucleotide saturation curves with ATP and CTP were performed on the (AT-C) 2 R 3 , (D236A-C) 2 R 3 , and (D236A-C)(AT-C)R 3 holoenzymes at onehalf the [Asp] 0.5 (Fig. 4). This aspartate concentration was selected because the nucleotide effects are enhanced at low aspartate concentrations (29). As was expected from the previously published results (13), the (D236A-C) 2 R 3 holoenzyme was not heterotropically activated by ATP and only slightly heterotropically inhibited by CTP. The heterotropic effects of ATP and CTP on the (AT-C) 2 R 3 holoenzyme were very similar to those observed for the wild-type holoenzyme (16). For the (D236A-C)(AT-C)R 3 hybrid holoenzyme, the heterotropic effects were a little less than half of that observed for the wildtype holoenzyme (see Table II). ATP activated the hybrid 150%, whereas the residual activity in the presence of CTP was 35%.
The Effect of PALA on the Mutant Holoenzymes-For wildtype aspartate transcarbamoylase at saturating concentrations of carbamoyl phosphate and subsaturating concentrations of aspartate, low concentrations of the bisubstrate analog PALA are able to substantially activate the enzyme. This activation is because of the ability of PALA to bind to a substoichiometric number of active sites and shift these enzyme molecules from the T to the R state. Once the enzyme has been converted to the R state the active sites that do not have PALA bound have enhanced activity and aspartate affinity.
As opposed to the wild-type enzyme, PALA is unable to activate the (D236A-C)(AT-C)R 3 holoenzyme at saturating carbamoyl phosphate and low levels of aspartate (data not shown). The inability of PALA to activate the (D236A-C)(AT-C)R 3 holoenzyme is consistent with the substantially reduced aspartate cooperativity of the hybrid.
Small Angle X-ray Scattering Experiments-To obtain direct structural data on the alterations to aspartate transcarbamoylase when the link between Asp-236c and Lys-143r was broken, small angle x-ray scattering was employed on Beamline 4-2 at the Stanford Synchrotron Research Laboratory.
Shown in Fig. 5A are the x-ray scattering curves for the native D236A and wild-type holoenzymes. As has been previously reported for the wild-type enzyme (30), the addition of PALA induces a significant alteration in the SAXS pattern of the unligated wild-type enzyme. PALA induces a major increase in scattering intensity and a shift in both the first subsidiary maximum and minimum. This alteration in the SAXS pattern induced by the binding of PALA is characteristic of the quaternary conformational change between the T and R states of the wild-type enzyme.
The SAXS pattern of the unligated native D236A holoenzyme is distinctly different from that of either the unligated or PALA-ligated wild-type holoenzyme. The SAXS pattern of the unligated native D236A holoenzyme exhibits both an increase in scattering intensity and shifts in both the first subsidiary maximum and minimum, however, these shifts are not as large as observed for the wild-type enzyme in the presence of PALA.
The SAXS pattern observed for the unligated native D236A holoenzyme could arise in two possible ways. First, the unligated native D236A holoenzyme could be in a new structural state different from either the unligated or PALA-ligated structures of the wild-type enzyme. Second, the mutation at Asp-236 could have altered the T to R equilibrium in such a fashion that a significant amount of the two structures are observed at equilibrium. If a mixture of T and R states was present the SAXS pattern would correspond to a weighted average of the SAXS patterns of the two independent structures. We have attempted to match the structure of the unligated native D236A holoenzyme using various mixtures of wild-type T state with wild-type R state, as well as wild-type T state and D236A R state. However, these attempts have not produced a match to the observed pattern for the unligated native D236A holoenzyme. Therefore, we believe that the unligated D236A holoenzyme is in a new structural state unlike either the T or R states of the wild-type enzyme. However, we consider these results tentative because of the relatively high noise level in the current set of SAXS patterns.
When PALA is added to the native D236A holoenzyme there is an alteration in the scattering pattern. In fact, the pattern for the native D236A holoenzyme is very similar to that observed for the wild-type enzyme in the presence of PALA, suggesting that the R state of the two enzymes is very similar in quaternary structure.
The SAXS pattern of the (D236A-C)(AT-C)R 3 holoenzyme in the absence of ligands is different from the corresponding patterns of the wild-type and native D236A holoenzymes (see Fig.  5B). The pattern is shifted toward a R structure but not as much as the native D236A holoenzyme. Again, in this case the SAXS curve for the (D236A-C)(AT-C)R 3 hybrid could not be reconstructed by a combination of T and R states. When PALA is added to the (D236A-C)(AT-C)R 3 holoenzyme the SAXS pattern is very similar to that observed for the wild-type and native D236A holoenzymes in the presence of PALA, suggesting that the R state of all these enzymes is very similar. These data suggest that three Asp-236c/Lys-143r interactions provide some T-state stabilization. DISCUSSION A comparison of the x-ray structures of the T and R states of aspartate transcarbamoylase reveal a series of interdomain and intersubunit interactions that appear to stabilize these two alternate functional and structural states of the enzyme (9)
(see Fig. 1). Site-specific mutagenesis has been used extensively to investigate the functional importance of many of the interdomain and intersubunit interactions observed in the T and R state x-ray structures (1,11,(31)(32)(33)(34). Two sets of interactions have been found to be important for the stabilization of the T state of the enzyme. The best characterized is the interaction between Glu-239c1 with both Lys-164c4 and Tyr-165c4 (11,12,35). When Glu-239c is replaced by Gln, the resulting enzyme exhibits full activity, no cooperativity, enhanced affinity for substrates, no activation by ATP, and no inhibition by CTP (11). Based upon functional studies, the native E239Q holoenzyme is functionally locked in the R state. Structural studies using SAXS have also been used to characterize the native E239Q holoenzyme (12). In the absence of ligands, the native E239Q holoenzyme is in a new structural state inconsistent with either the T or R structures of the wild-type enzyme (12). The position of the SAXS pattern for the E239Q holoenzyme suggests that the loss of the intersubunit interactions destabilizes the T state and the unligated enzyme exists in a new structural state between the T and R states of the wild-type enzyme. Because the intersubunit interactions involving E239Q only exist in the T state of the enzyme, this suggests that the structural intermediate observed in the x-ray scattering of the E239Q holoenzyme may be a structural intermediate in the T to R transition of the wild-type enzyme. This notion is supported by the fact that PALA is able to convert the unligated structure of the E239A holoenzyme into a structure that is virtually identical to that observed with the PALAligated wild-type enzyme (12).
A study utilizing a series of hybrid holoenzymes, in which one to five of the catalytic chains had the E239Q mutation, revealed that three of the six intersubunit interactions are sufficient to stabilize the enzyme in the T state, thereby allowing the retention of homotropic cooperativity and heterotropic activation and inhibition (16,36).
Another intersubunit interaction that stabilizes the T functional state of aspartate transcarbamoylase is between Asp-236c1 and Lys-143r4. The replacement of Asp-236 by alanine results in a mutant holoenzyme (D236A) that had remarkably similar properties to the E239Q holoenzyme, full activity, no cooperativity, enhanced affinity for substrates, no activation by ATP, and little inhibition by CTP (13). To more fully establish the importance of the intersubunit interactions between Asp-236c1 and Lys-143r4 for the function of aspartate transcarbamoylase and to compare the loss of this catalytic-regulatory intersubunit interaction to the catalytic-catalytic intersubunit interaction involving Glu-239, we report here the characterization of a hybrid enzyme that has only three of the six possible catalytic-regulatory intersubunit interactions involving Asp-236. In addition, the structural consequences of the replacement of Asp-236 by alanine were also investigated in both the native D236A holoenzyme and the hybrid by small-angle x-ray scattering.
To isolate the hybrid holoenzyme in which one catalytic subunit had the D236A mutation and the other had the wildtype amino acid, Asp, at position 236, a modified version of the wild-type catalytic chain with six additional Asp residues appended to the C-terminal was used. We have previously shown that a holoenzyme consisting of two of these modified wild-type catalytic subunits (AT-C) and wild-type regulatory subunits, (AT-C) 2 R 3 , exhibited kinetic and structural characteristics identical to that of the corresponding wild-type holoenzyme lacking the Asp tail (16). Furthermore, the additional negative charge on the AT-C catalytic subunits is sufficient to resolve by chromatography the three holoenzyme species produced by reconstitution of AT-C and D236A-C catalytic subunits with wild-type regulatory subunits (R) (see Fig. 2).
Comparison of the (D236A-C)(AT-C)R 3 hybrid holoenzyme with the wild-type and native D236A holoenzymes revealed that the hybrid exhibits similar catalytic activity to both species. Although the [Asp] 0.5 is diminished for the hybrid compared with the native D236A holoenzyme, the [Asp] 0.5 of the (D236A-C)(AT-C)R 3 hybrid holoenzyme is still about 3-fold higher than the wild-type enzyme. In addition, the (D236A-C)(AT-C)R 3 hybrid holoenzyme exhibits slight aspartate cooperativity, however, the low [Asp] 0.5 of the hybrid holoenzyme makes the accurate kinetic determination of residual cooperat-  8.3 These data were determined from ATP and CTP saturation curves (Fig. 4)  ivity difficult. Therefore, the slight cooperativity observed for the (D236A-C)(AT-C)R 3 hybrid indicates that three of the six Asp-236c/Lys-143r interactions are sufficient for stabilization of the T state of the enzyme. The lower [Asp] 0.5 of the hybrid and native D236A enzymes is most likely because of a slight alteration in the position of the critical 240s loop in the R state.
As opposed to the (D236A-C) 2 R 3 holoenzyme, the (D236A-C)(AT-C)R 3 hybrid holoenzyme was activated by ATP and inhibited by CTP, although the extent of the activation and inhibition were reduced compared with the wild-type holoenzyme. The activation by ATP and the inhibition by CTP of the (D236A-C)(AT-C)R 3 hybrid holoenzyme indicates that three of the six Asp-236c to Lys-143r interactions are sufficient to restore heterotropic interactions and the ability of the hybrid holoenzyme to undergo a T to R transition.
To obtain direct structural data on the conformation of the native D236A holoenzyme and the (D236A-C)(AT-C)R 3 hybrid, small angle x-ray scattering (SAXS) experiments were performed. As a control, the SAXS curves were also recorded for the wild-type holoenzyme. The native D236A holoenzyme without ligands exhibits a scattering pattern that is different from the T and R patterns of the wild-type enzyme (see Fig. 5). The SAXS pattern of the native D236A holoenzyme without ligands suggests that this enzyme is in a new structural state, as no combination of the wild-type T and R state patterns fit the native D236A holoenzyme scattering pattern, within error. When PALA is added to the native D236A holoenzyme, the SAXS pattern changes and is very similar to that of the PALAligated wild-type enzyme suggesting that the R state structures of the two enzymes are very similar.
The SAXS pattern for the unligated (D236A-C)(AT-C)R 3 hybrid is different from either the unligated wild-type or the native D236A holoenzyme. However, the SAXS pattern of the hybrid is much more similar to the unligated wild-type than the native D236A holoenzyme, providing additional support for the hypothesis that three of the six Asp-236c/Lys-143r interactions provide significant T state stabilization to the enzyme. Addition of PALA to the (D236A-C)(AT-C)R 3 hybrid gives a SAXS pattern very similar to the PALA-ligated wild-type pattern, suggesting that the R states of these enzymes are virtually identical.
We have previously investigated another set of intersubunit interactions between Glu-239c1 and both Lys-164c4 and Tyr-165c4. The native D236A and E239Q holoenzymes as well as the hybrid holoenzymes, containing one wild-type and one mutant catalytic subunit, have remarkably similar properties. For the unligated native E239Q holoenzyme, the SAXS pattern is very similar to that of the unligated native D236A holoenzyme reported here. Furthermore, the kinetic characterization and the SAXS pattern of the (E239Q-C)(AT-C)R 3 and (D236A-C)(AT-C)R 3 hybrid holoenzyme are quite similar. In both cases the hybrid has restored cooperativity and heterotropic interactions, and the SAXS patterns of the hybrids are very T-like. Both sets of data suggest that three of the six intersubunit interactions add significant stabilization to the T state of the enzyme.
The saturation of only a small number of active sites by substrates (or substrate analogs) to aspartate transcarbamoylase is sufficient to induce the allosteric transition (37). The binding of aspartate to enzymes previously saturated with carbamoyl phosphate induces the critical closure of the domains of the catalytic chains (38). However, as we have previously proposed this domain closure in one catalytic chain cannot occur without some change in the quaternary structure of the enzyme because of steric interference of this domain closure (39). Thus, the elongation of the enzyme is a necessary part of the allosteric transition of aspartate transcarbamoylase. The elongation of the molecule requires the breaking of the T state intersubunit interactions involving both Asp-236 and Glu-239. When the side chain of either of these residues is replaced by noncharged substitutes, the net results are the inability of the enzyme to exist in the T state and the formation of a new structural state that is neither T nor R. Because the interactions involving both Glu-239 and Asp-236 are lost during the wild-type T to R transition, it is reasonable to speculate that the structures of the unligated D236A and E239Q holoenzymes are intermediates in the pathway of the wild-type T to R transition. The fact that the E239Q holoenzyme can be shifted to the R state with only the addition of carbamoyl phosphate suggests that the final conformational changes necessary to create the R state may be partially coupled to the major quaternary conformational change. Additional SAXS experiments are currently underway to better establish the nature of the allosteric transition for both the wild-type and mutant versions of aspartate transcarbamoylase.