The First High pH Structure of Escherichia coli Aspartate Transcarbamoylase

Abstract

The activity and cooperativity of Escherichia coli aspartate transcarbamoylase (ATCase) vary as a function of pH, with a maximum of both parameters at approximately pH 8.3. Here we report the first X-ray structure of unliganded ATCase at pH 8.5, to establish a structural basis for the observed Bohr effect. The overall conformation of the active site at pH 8.5 more closely resembles the active site of the enzyme in the R-state structure than other T-state structures. In the structure of the enzyme at pH 8.5 the 80’s loop is closer to its position in R-state structures. A unique electropositive channel, comprised of residues from the 50’s region, is observed in this structure, with Arg54 positioned in the center of the channel. The planar angle between the carbamoyl phosphate and aspartate domains of the catalytic chain is more open at pH 8.5 than in ATCase structures determined at lower pH values. The structure of the enzyme at pH 8.5 also exhibits lengthening of a number of interactions in the interface between the catalytic and regulatory chains, whereas a number of interactions between the two catalytic trimers are shortened. These alterations in the interface between the upper and lower trimers may directly shift the al- losteric equilibrium and thus the coopera- tivity of the enzyme. Alterations in the elec- tropositive environment of the active site and alterations in the position of the cata- lytic chain domains may be responsible for the enhanced activity of the enzyme at pH 8.5.

INTRODUCTION

The Bohr effect was first observed for hemoglobin by Christian Bohr in 1904.¹ Bohr observed that an increase in blood carbon dioxide levels accompanied by a decrease in pH, caused hemoglobin to bind oxygen with less affinity. Today, the Bohr effect more commonly refers to alterations in substrate affinity or enzyme activity as a function of pH, and has been studied extensively in a variety of allosteric proteins and enzymes such as hemoglobin.^2,3

Escherichia coli aspartate transcarbamoylase (EC 2.1.3.2, ATCase) is an allosteric enzyme whose activity is strongly modulated by pH, exhibiting maximum activity and cooperativity at pH 8.3.⁴ The enzyme exhibits homotropic cooperativity for aspartate,⁵ and is heterotropically regulated by the end-products of the pyrimidine nucleotide pathway CTP,⁵ as well as, UTP in the presence of CTP.⁶ ATP, the end-product of the parallel purine pathway, activates the enzyme.⁵ In the absence of substrates, the enzyme is predominately in the low-affinity, low-activity T state, whereas the enzyme with both substrates or the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA), is predominately in the high-affinity, high-activity R state.⁷ The T to R transition involves an expansion of the molecule by 11 Å along the threefold axis, a rotation of the catalytic subunits by 158 about the molecular threefold axis, and a rotation of the regulatory subunits by 108 about their respective twofold axes.^8,9

The active sites of ATCase are shared between adjacent catalytic chains in the trimer.^10–12 On the binding of carbamoyl phosphate (CP) local conformational changes occur, forming the high-affinity binding pocket for Asp due to the repositioning of the 50’s loop (residues 50–55) and the movement of the 80’s loop (residues 73–88) from the adjacent chain into posi- tion to bind the CP molecule.¹³ On the binding of Asp to the enzyme CP complex, the two domains of the cata- lytic chain undergo a domain closure, bringing CP and Asp into close proximity for reaction.¹⁴

The influence of pH on ATCase has been studied by analysis of proton binding, substrate binding, and conformational transitions.¹⁵ These studies evaluated the net changes in the state of ionization of the protein and substrate analogues.¹⁵ Isotope trapping studies revealed that at least four ionizable side chains are involved in sub- strate binding and catalysis.¹⁶ Two residues with pKa values of 7 and 9 must be protonated for the binding of CP and Asp, respectively. The catalytically productive form of the enzyme CP complex has a group with a pKa of 7.0 protonated and a group with a pKa of 9.1 deprotonated. When studying the pH effect on the kinetic and regulatory properties of ATCase, Pastra-Landis et al.⁴ proposed that there is a residue essential for cooperativity at or near the Asp binding site with a pKa of 8.5. This study also suggested that the pH-dependent shift in the Asp saturation curve is largely because of changes involving intersubunit salt bridges. Finally, a detailed study of the Bohr effect in ATCase was performed using calorimetry, potentiometry, and spectrophotometry to analyze the coupling between the binding of protons and substrate analogues.^15,17 This work identified two residues with pKa values of 6.3 and a third residue with a pKa of 9.1 that play a critical role in the Bohr effect of ATCase.¹⁸

Extensive studies of the three-dimensional structure of E. coli ATCase at low pH (5.9) in the presence and absence of substrate/substrate analogues have been reported,^8,19,20 as well as additional structural studies of the enzyme at pH 7.^21–24 However, no structure of the unliganded enzyme has been determined at a pH where the enzyme exhibits maximal activity and cooperativity (pH 8.3), due to difficulties in crystallizing the enzyme under these conditions. Here, we report the first X-ray structure of unliganded E. coli ATCase at pH 8.5. These data shed light on the structural basis for how alterations in pH influence the catalysis and cooperativity of the enzyme.

Materials and Methods

Materials

Agar, ampicillin, 2-mercaptoethanol, polyethyleneglycol 4000, potassium dihydrogen phosphate, sodium ethylenediaminetetracetic acid, sodium acetate, uracil were obtained from Sigma Chemical Co. (St. Louis, MI). Q-Sepharose Fast Flow resin was obtained from Amersham Pharmacia Biosciences. Casamino acids, yeast extra and tryptone were obtained from Difco (Detroit, MI). Sodium dodecyl sulfate was purchased from Bio-Rad Laboratories. Enzyme grade ammonium sulfate, Tris, electrophoresis grade acrylamide, agarose, were purchased from ICN Biomedicals (Costa Mesa, CA). Crystal cryo-mountig loops and siliconized cover slides were obtained from Hampton Research (Laguna Niguel, CA).

Methods

Expression and purification of the enzyme

E. coli ATCase was overexpressed utilizing strain EK110425 containing the plasmid pEK152.²⁶ The overexpression, isolation, and purification of the enzyme were performed as described previously.²⁵ The concentration of enzyme was determined by absorbance measurements at 280 nm with an extinction coefficient of 0.59 (mg/ml)⁻¹ cm⁻¹.²⁷ The purity of the enzyme was checked by SDS-PAGE²⁸ and nondenaturing PAGE.^29,30

Crystallization and freezing of crystals

ATCase was crystallized by the hanging drop vapor dif- fusion. The enzyme solution, at 10 mg/mL, was mixed 50%/50% (v/v) with a solution of crystallization buffer (100 mM Tris, 200 mM Li₂SO₄, 25% PEG3300 (w/v) pH 8.5), and then equilibrated against 1 mL of crystallization buffer. Crystals grew to average dimensions of 0.3 × 0.3 × 0.1 mm³ within 5 weeks. Crystals were transferred into a cryoprotectant composed of 20% glycerol in crystallization buffer for ~1 min before the crystals were frozen in liquid nitrogen.

X-ray data collection and processing

The data for the structure in the ATCase were collected using a Rigaku R-axis IV++ detector while X-rays were generated using a Rigaku RU-200 rotating-anode generator operating at 50 kV and 100 mA at the Crystallographic Facility in the Chemistry Department of Boston College. The diffraction data were integrated, scaled and averaged using HKL2000.³¹

Structural Refinement

The initial model for the high pH structure was derived from the coordinates of a mutant version of ATCase (K244N) (PDB code 1SKU) which crystallized at pH 7 in the same space group (H3) and with similar unit cell dimensions (a = b = 125.67 c = 198.20).³² Before refinement with CNS,³³ all the waters and ligands were removed. After initial rigid body, simulated annealing, minimization, and B-factor refinement the initial maps were inspected. The 80’s loop of the catalytic chain was found to be disordered. Several rounds of manual rebuilding, using XtalView³⁴ and refinement were performed to correct the structure.

Waters were added to the structure on the basis of Fo - Fc electron density maps at or above the 2.5σ level using COOT³⁵ and subsequently refined with REFMAC.³⁶ Waters were retained only when they could be justified by hydrogen bonds. The model was checked for errors using PROCHECK.³⁷ The details of data collection, processing, and refinement are provided in Table I.

Table I.

X-ray Data Collection and Refinement statistics for the ATCase pH 8.5 Structure

Data collection
	Space Group	H3
	Cell Dimensions:
		a = b, c (Å)	129.78, 197.74
		α, β, γ (°)	90, 90, 120
	Resolution (Å)	50.00−2.80
	Rmergea(%)	0.106 (0.329)
	Average (I/σ)	5.8 (2.1)
	Completeness (%)	99.7 (99.0)
	Total Reflections	102,066
	Unique reflections	27,528
	Redundancy	3.71 (3.45)
Refinement
	Resolution (Å)	50.00−2.80
	Reflections	80,493
	Rfactor/Rfree	0.215/0.247
RMS deviations:
	Bonds (Å)	0.014
	Angles (°)	0.83
	Mean B value (Å2)	40.8
	Total number waters	213

Values in parentheses are for the highest resolution shell

^a

$${\mathrm{R}}_{\text{merge}}=\frac{{\displaystyle \sum |{\mathrm{I}}_{\text{hkl}}-\overline{{\mathrm{I}}_{\text{hkl}}}|}}{{\displaystyle \sum \overline{{\mathrm{I}}_{\text{hkl}}}}}$$

Method of calculating pKa’s of residues

Residue pK_a values were calculated using MCCE³⁸ which utilizes Delphi as the Poisson-Boltzmann equation solver.³⁹ To include all residues in the shared active site, the calculations were performed on the combination of one C1 and one C2 catalytic chain. The MCCE program was run in quick mode with the dielectric constant of the protein set to four. Analysis of the final conformation of the protein side chains of the T_{low_apo} and the T_{high apo} structures revealed that the side chains of active site residues varied less than 1.5 Å from their original positions.

RESULTS AND DISCUSSION

The study presented here examines how pH influences the structure of ATCase and to what degree this is responsible for the observed Bohr effect of ATCase. The most noticeable effect of pH on the function of ATCase is that the activity of the enzyme increases dramatically as the pH increases from 6.1 to 8.8. The maximal observed velocity of ATCase at pH 8.8 is almost 11-fold higher than the maximal velocity at pH 6.1.⁴ In addition to this dramatic increase in activity, ATCase lacks cooperativity at low pH (Hill coefficient = 1 at pH 6.14). Cooperativity increases as a function of pH with the Hill coefficient attaining a maximal value at pH ~8.5.⁴

Three-dimensional structure of E. coli ATCase at pH 8.5

ATCase crystallized at pH 8.5 in the space group H3 rather than the more common P321 space group observed for ATCase crystals. The unit cell dimensions of the crystals of ATCase grown at pH 8.5, in the H3 space group, T_{high_apo}, (a = b = 129.78 A, c = 197.74 A) are similar to the unit cell dimensions of the few other T- state crystals of ATCase in the H3 space group. Two of these structures in the H3 space group are of the wild- type enzyme with substrates or substrate analogs in the active site (PDB entries: 1ROC²⁴ and 2AIR⁴⁰), whereas two were for the P268A (PDB entry 1EZZ⁴¹) and the K244N (PDB entry 1SKU³²) mutant versions of ATCase in the absence of substrates or substrate analogues. The similarity of the unit cell dimensions of the T_{high_apo} crystals reported here, as compared with these known T- state structures in the same space group grown at pH values of seven or less, suggested that the crystals grown at pH 8.5 were in the T-quaternary structure. Therefore, the initial model used for the refinement of the structure was the T-state structure of the K244N mutant ATCase in the H3 space group (PDB code 1SKU).³² The data for the T_{high_apo} were refined to a R_factor and R_free of 21.5% and 24.7%, respectively, with 213 water molecules in the asymmetric unit.

Quaternary structure

To evaluate the quaternary structure of the enzyme, the vertical separation along the threefold axis between the center of mass of the upper and lower catalytic subunits were determined and compared with previously determined structures of ATCase in the T and R states. The vertical separation for ATCase in the T and R states was 45.6 Å and 56.6 Å, respectively.⁴² The difference between the R- and T-state vertical separations corresponds to the vertical expansion of the enzyme observed in the T to R transition (56.6 − 45.6 = 11 Å). For the T_{high_apo} structure the vertical separation was 45.2 Å, similar to the vertical separation of the T state of the enzyme determined at other pH values. These data indicate that the T_{high_apo} structure reported here is in the T-quaternary conformation.

Tertiary structure of the catalytic chain of ATCase at high pH

To better understand the influence of pH on the homotropic cooperativity of ATCase the catalytic chains in the T_{high_apo} structure were compared, after superposition with CNS,33 to the catalytic chains of the T_{low_apo} and R_{low_pala} structures (see Fig. 1). The RMS deviation of the α-carbon atoms between the C1 catalytic chain of the T_{high_apo} and T_{low_apo} structures was 1.33 Å . Shown in Figure 1(A) is the structure of one catalytic chain (C1) with the portion of the 80’s loop of the adjacent catalytic chain (C2) that donates residues into the active site. On the tertiary level, the two structures are very similar with the exception of the 80’s loop from residues 73 to 88 (C1 and C2) and a small region of the 240’s loop, which are both clearly different.

Figure 1.

A: Comparison of the C1 chain and the 80’s loop of the C2 chain of the T_{high_apo} with corresponding portions of the T_{low_apo} structure. B: Comparison of the C1 chain and the 80’s loop of the C2 chain of the T_{high_apo} with corresponding portions of the R_{low_pala} structure. The width of the tube is proportional to the RMS deviation of the a-carbon atom positions. Also shown is the 80’s loop from the adjacent catalytic chain that contributes residues to the active site (80’s Loop*). C: Stereoview of a superposition of the a-carbon atoms of the T_{high_apo} (blue), T_{low_apo} (gray), and the R_{low_pala} (green) structures. Parts A and B were prepared using CHIMERA,⁴³ whereas Part C was prepared using PyMol.⁴⁴

Shown in Figure 1(B) is a similar comparison between the T_{high_apo} and R_{low_pala} structures. The RMS deviation of the α-carbon atoms between the C1 catalytic chain of the T_{high_apo} and R_{low_pala} structures was 2.61 Å. The region that shows the largest difference between the two structures is the 240’s loop. These data indicate that the tertiary structure of the catalytic chains of the T_{high_apo} structure are in a T-like conformation. However, in the T_{high_apo} structure the 80’s loop, which is critical for the initiation of the T to R transition, is in a significantly different position than in the T_{low_apo} structure [Fig. 1(A)]. The overall RMS deviation including both α-carbon and side chain atoms between the 80’s loop of the T_{high_apo} catalytic chain and the R_{low_pala} and T_{low_apo} structures were 5.58 Å and 4.69 Å, respectively, whereas the corresponding RMS deviation between the T_{low_apo} and R_{low_pala} structures was 11.45 Å [Fig. 1(C)]. The B-factors for the 80’s loop residues in the T_{high_apo} struc-ture are similar to those in other structures determined at lower pH. The conformation of the 80’s loop in the T_{high_apo} structure is also closer to R_{neutral_pala} structure than to the T_{neutral_ca} structure. In addition to a major alteration in the position of the 80’s loop, there are also smaller but significant alterations in portions of the 50’s and 240’s loops. These loop alterations may account for changes in substrate affinity and activity, since the 50’s and 80’s loops are involved in the binding of CP and the 80’s and 240’s loops are involved in the binding of Asp.

Tertiary structure of the regulatory chain of ATCase at high pH

To further understand how pH can influence the properties of ATCase, the T_{low_apo}, and the T_{high_apo} regulatory chains were compared. The RMS deviation of α-carbons of the T_{high_apo} and the T_{low_apo} regulatory chain was 2.8 Å. There were some small but significant differences in the tertiary structure between the two structures in the 50r’s loop and in the 130r’s loop of the regulatory chains. The electrostatics in the region around Arg130r, Asn132r, and Asp133r of the T_{high_apo} structure exhibited large shifts compared with structures determined at lower pH values. The differences in the 50r’s region are due to changes in the architecture of the allosteric binding site. More specifically, a comparison of the allosteric binding site of the T_{high_apo} and T_{low_apo} structures revealed that there is a hydrogen-bonding network present in the Thigh_apo structure that is not present in the T_{low_apo} structure. Residues Asp19r, His20r, Ser50r, and Lys56r are engaged in a hydrogen-bonding network in the T_{high_apo} structure with the side chain of nearby Glu52r. His20r in the T_{low_apo} structure interacts only with the backbone of Glu52r and Lys56r, but does not interact with Asp19r or Ser50r as observed in the T_{high_apo} structure. In the T_{low_apo} structure, Lys56r only interacts with Asp19r. The interactions involving Lys56r in the T_{high_apo} structure can be used to help explain the important functional role of this residue.⁴⁵

As opposed to most structures of the enzyme at low pH, the N-terminus (residues 1r–13r) of the T_{high_apo} structure was well defined in electron density maps at 1.8σ and appeared to be in a unique conformation that may influence the binding of ATP and CTP, as more hydrogen bonding donors are facing inward toward the phosphate binding pocket.

The active site of the Thigh_apo structure

The 2Fo - Fc electron density map of the C1 active site of the T_{high_apo} structure is shown in Figure 2(A). The side chains in the active site were well ordered even though there was no substrate or substrate analogues present. The active site of the T_{high_apo} structure is more similar to the active site in the R state, than any other T-state structures. As comparison of the T_{low_apo} structure there where significant changes in the side chain positions of Arg105, Arg229, Lys232, as well as residues of the adjacent 80’s loop. To better understand the positional changes that occur to accommodate substrate binding, the T_{low_CTP_CP} structure was compared with the T_{high_apo} structure. The conformation of the 50’s loop region of the T_{high_apo} structure, as compared with the T_{low_CTP_CP} structure [Fig. 2(B)] ideally positions the CP domain to interact with the carbonyl oxygen and nitro- gen of CP. More specifically, the distance from the carbonyl oxygen of CP to the nitrogen H-donor of Arg54 was 0.8 Å less in the T_{high_apo} as compared with the T_{low_CTP_CP} structure. The same structural comparison revealed that the distance from Arg105 to both the phosphate oxygen and carbonyl oxygen was 0.6 Å less. In contrast, Ser52, Thr53, and Thr55 as well as Ser80 from the adjacent catalytic chain were in different conformations in the T_{high_apo} and T_{low_CTP_CP} structures [Fig. 2(B)]. The T_{low_apo} was also compared with the T_{high_apo} structure and both the 80’s and 50’s regions were in different conformations. The T_{high_apo} structure is not intermediate between the T_{low_CTP_C}P and T_{low_apo} structures, but rather is in a unique conformation.

Figure 2.

A: The 2Fo - Fc electronic density map of active site of the T_{high_apo} structure contoured at 1.8σ with the refined coordinates overlaid. B: Overlay of active site of the T_{high_apo} structure (gray) and the T_{low_CTP_CP} structure (black). The interactions between the CP and enzyme in the T_{low_CTP_CP} structure are shown as dotted lines. Superposition of the structures was performed using SEQUOIA⁴⁶ and the figure was prepared with POVScript1.⁴⁷

To evaluate the electrostatics of the active site, the electrostatic potentials mapped onto the molecular surface of T_{high_apo}, T_{low_apo}, and T_{low_CTP_CP} structures were compared. As seen in Figure 3(A), the T_{high_apo} structure has a unique positively charged “cleft” or “channel” consisting of residues from the 50’s region with Arg54 positioned in the center of the channel. In the T_{high_apo} structure, the previously seen T-state stabilizing interaction between Arg54c1* and Glu86c2 is not observed. In the T_{high_apo} structure, Arg54 forms an interaction with Ser52, stabilizing the observed alternate conformation of the 50’s region, and an interaction with Asn78c2 stabilizing the observed alternate conformation of the 80’s loop [Fig. 4(A)].

Figure 3.

Electrostatic potentials mapped on the molecular surface of active site of the (A) T_{high_apo} structure, (B) T_{low_apo} structure, and (C) T_{low_CTP_CP} structure in the range ±15 kT/e. The positions of Arg54, His134, Arg105, Arg229, and Gln231 and Lys83 and Lys84 from the adjacent chain are shown. Electrostatic potentials were calculated using APBS.⁴⁸ The dielectric of the protein and the solvent were set to 4 and 80, respectively. This figure was prepared with PyMol⁴⁴ and the APBS tools plug-in employing standard charges on the amino acid side chains except for those indicated in Table II. For these amino acids the charges were calculated at pH 7 using the indicated pK_a values in Table II.

Figure 4.

The active site architecture of the (A) T_{low_apo} and (B) T_{high_apo} structures. Residues from C1 chain are in gray and residues from C2 chain are in magenta. An asterisk after the residue number indicates it is from the C2 chain. This figure was drawn with POVScript1.⁴⁷

For comparison, Figure 3(B) shows the electrostatic potential mapped on the molecular surface of the T_{low_apo} structure. The CP domain portion of the active site in the T_{high_apo} structure is much more electropositive than the corresponding portion of the active site of the T_{low_apo} structure. In addition, the 50’s region is in a more closed conformation in the T_{low_apo} structure as compared with the T_{high_apo} structure [compare Fig. 3(A,B)]. In the case of the T_{low_CTP_CP} structure, both the CP and Asp domain exhibit a diminished positive electrostatic surface potential as compared with the T_{high_apo} structure. However, in the T_{low_CTP_CP} structure, the CP and Asp domains are in a more closed conformation than the T_{high_apo} and T_{low_apo} structures [compare Fig. 3(C) with Fig. 3(A,B)]. The difference in the electrostatics of the active site of ATCase between the structures determined at low and high pH is induced by the observed local conformational changes of both the 50’s and the 80’s loops. Upon the binding of CP, the 50’s region undergoes structural alterations that are critical for the formation of the high-activity, high-affinity Asp binding site.¹³ The positive electrostatic environment of the active site will promote neutralization of negatively charged CP on binding and make the phosphate a better leaving group. It is tempting to speculate that the electropositive channel observed in the T_{high_apo} structure provides a path for easy entry of CP into the active site. Since the catalytic activity of the enzyme is highly dependent on Arg54,^49,50 the repositioning of the Arg54 side chain may contributes to the Bohr effect of ATCase.

The structural effect of pH on the active site architecture as observed in the T_{high_apo} structure is shown in Figure 4. In the T_{high_apo} structure, there is a unique hydrogen-bonding network involving residues Arg105, Thr55, Ser52, Arg54, and Asn78 of the 80’s loop of the adjacent chain [Fig. 4(B)]. Such an intricate hydrogen- bonding network is not observed in other ATCase structures determined at lower pH [see Fig. 4(A)]. In the Asp domain of the T_{high_apo} structure, there is a hydrogen-bonding network involving residues Gln231, Arg229, Lys232, Asp271, Tyr240, and Lys84 of the adjacent catalytic chain [Fig. 4(B)] that is not observed in the T_{low_apo} structure [Fig. 4(A)]. The interactions observed in the active site of the T_{high_apo} structure are similar to those observed in the R-state structure of the enzyme bound with an analogue of CP, phosphonoacetylamide (R_{D236_PAM}).⁵¹ These observations further support the notion that the active site of the Thigh_apo structure has certain characteristics of the R-state structure.

The position of 80’s loop, stabilized by interactions between Lys232c1 and Lys84c2 as well as between Arg54c1 and Asn78c2, in the T_{high_apo} structure con- tributes to a more open conformation of the active site cavity [Fig. 4(B)], rendering the active site cavity more solvent accessible. The enhanced electropositive environment of the active site in the T_{high_apo} structure (see Fig. 3) also provides a more complementary electrostatic interaction for CP.

Relative positions of the catalytic chain domains

The planar angle⁵² between the CP and Asp domains was determined to quantify and compare the degree of openness of the active site of the T_{high_apo} structure relative to the active sites of other T-state and R-state structures. The planar angles between the CP and Asp domains of the T_{high_apo} structure were 139.38 and 137.58 for the C1 and the C6 catalytic chains, respectively. In contrast the planar angles between the CP and Asp domains of the T_{low_apo} structure are more closed, with angles of 135.78 and 134.18 for the C1 and C6 chains, respectively. Since these planar angles are calculated using the center of mass of the two domains and a hinge point (α-carbon of residue 140), differences larger than 18 are significant. The planar angles between the CP and Asp domains of the T_{low_CTP_CP} structure were also more closed than in the T_{high_apo} structure (133.48 and 135.28 for the C1 and C6 chains, respectively). In addition, the product bound T structure, T_{neutral_ca}, has planar angles between the CP and Asp domains of 137.58 and 137.48 for the C1 and C6 chains, respectively, which is a more closed conformation than observed in the T_{high_apo} structure. In summary, the two domains of the catalytic chain in the T_{high_apo} structure are in a more open conformation than in any other structure at lower pH values. A more open conformation may not necessarily promote higher affinity, but the more positive electrostatic environment observed, combined with the increased solvent accessibility, may pro- vide better access of CP to the active site cavity.

Alterations in interchain interactions

To understand how changes in pH influence the inter- chain interactions of ATCase, the C1–R1 and C1–C4 interchain interactions of the T_{high_apo} and T_{low_apo} structures were compared. Most of the C1–R1 interactions were further apart in the T_{high_apo} structure than the corresponding interactions in the T_{low_apo} structure. For example, the interaction between Pro107c1 and Asn113r1 increased from 3.0 Å in the T_{low_apo} to 3.5 Å in the T_{high_apo} structure. The interaction between Glu109c1 and Asn113r1 increased from 3.0 Å in the T_{low_apo} to 3.6 Å in the T_{high_apo} structure and the interaction between Ser131c1 and Lys143r1 increased from 2.7 Å in the T_{low_apo} to 3.1 Å in the T_{high_apo} structure. A new interaction was also observed in the T_{high_apo} structure between Gln133c and Glu142r.

As opposed to the increase in distance of the interactions between the C1–R1 chains, the interchain interactions between the C1–C4 chains at high pH are shorter by 0.5–0.6 Å compared with the length of these interactions in the low pH structures. For example, the critical T-state stabilization interaction between Glu239c1 and both Tyr165c4 and Lys164c4 are both 2.65 Å in T_{high_apo} structure, compared with 3.17 Å and 3.24 Å in the T_{low_apo} structure. The shorter distances of these C1–C4 interactions suggest that they are stronger and that at high pH the T-state of the enzyme is more stable than at lower pH. The enhanced stabilization of the T-state at high pH may be responsible for the higher observed cooperativity.

Calculation of pKa values

The MCCE³⁸ program was used to investigate alterations in pK_a values because of the structural changes between the T_{low_apo} and the T_{high_apo} structures. Within the active site area there were significant shifts in pK_a values (>1.0 pK_a units) for four residues: His134c1, Lys232c1, Lys84c2, and Glu86c2 (see Table II). His134 has been proposed to act as a base accepting a proton from the amino group of Asp.⁵³ When His134 is replaced by Ala, the mutant enzyme exhibits a 20-fold reduction in activity as compared with the wild-type enzyme. In the T_{high_apo} structure, His134 is observed to help create the positive electrostatic channel in the “R-like” active site. The MCCE³⁸ calculations suggest that the pK_a of His134 is 1.8 units higher in the T_{high_apo} as compared with the T_{low_apo}. This increase would enhance the ability of His134 to accept protons. The pK_a of Glu86 in the T_{high_apo} structure was 8.5, 3.5 units higher than in the T_{low_apo} structure. This higher pK_a is within the pKa range of the group that must be protonated for binding of the substrates.¹⁶ The structural basis for this may be due to the formation of a salt link between Glu86c2 with Arg54c1. Also, the pK_a of Lys84c2 decreases by 2.5 units and the pK_a of Lys232c1 increases by 1.9 units. The decrease in the pK_a of Lys84c2 supports the previous suggestion that this residue may act as a base accepting a proton from the tetrahedral intermediate.⁵⁴ The alterations in the pKa values of these two Lys residues also have a dramatic effect on the shape of the Asp binding pocket [compare Fig. 3(B,C)].

Table II.

Comparison of calculated pK_a values for side chains in the T_{low_apo} and T_{high_apo} structures. See the Methods and Materials section for details of the pK_a calculations that were done using MCCE. Only these residues which had ΔpK_a values greater than one are shown.

Side Chain pKa Values
Residue	Tlow_apo	Thigh_apo	ΔpKa
Lys84c2	10.8	8.7	2.5
Lys232c1	10.0	11.9	1.9
His134c1	5.8	7.6	1.8
Glu86c2	5.0	8.5	3.5

SUMMARY

Analysis of the T_{high_apo} structure demonstrates that the pH influences homotropic cooperativity by strengthening the interactions between the interface between the upper and lower catalytic trimers (e.g., the C1–C4 interface and the symmetry related interfaces) and thereby shifting the allosteric equilibrium toward the T state. Analysis of the structure also suggests that the enhanced catalytic activity of the enzyme at pH 8.5 may be because of the observed alterations in the electrostatics of the active site as well as a more open conformation of the two domains of the catalytic chain. pH also influences the interactions between the catalytic and regulatory chains which could be responsible for the observed changes in heterotropic properties of the enzyme as a function of pH. To fully understand how the structural changes due to alterations in pH influence the homo- tropic and heterotropic properties of ATCase an X-ray structure of the enzyme in the R state at high pH would be extremely useful.

Abbreviations

ATCase

Escherichia coli aspartate transcarbamoylase holoenzyme

CP

carbamoyl phosphate

Asp

L-aspartate

PAM

phosphonacetamide

PALA

N-phosphonacetyl-L-aspartate

CA

N-carbamoyl-L-aspartate

C1/C6

the two catalytic chains of ATCase in the asymmetric unit of the crystal

R1/R6

the two regulatory chains of ATCase in the asymmetric unit of the crystal

C/R

the catalytic and regulatory chain interface

T_{high_apo}

the T-state X-ray structure of unliganded ATCase determined at pH 8.5 and reported here

T_{low_CTP}

the T-state X-ray structure of ATCase in the presence of CTP determined at pH 5.8 (PDB code 1ZA1)

T_{low_CTP_CP}

the T-state X-ray structure of ATCase in the presence of both CTP and CP determined at pH 5.8 (PDB code 1ZA2)

T_{low_apo}

the T-state X-ray structure of unliganded ATCase determined at pH 5.8 (PDB code 6AT1)

R_{low_pala}

the R-state X-ray structure of ATCase in the presence of PALA determined at pH 5.8 (PDB code 1D09)

T_{neutral_ca}

the T-state X-ray structure of ATCase in the presence CA and phosphate determined at pH 7.0 (PDB code 1R0C)

R_{neutral_pala}

the R-state X-ray structure of ATCase in the presence of PALA determined at pH 7.0 (PDB code 1Q95)

RMS

root mean square