Structural similarity between ornithine and aspartate transcarbamoylases of Escherichia coli: characterization of the active site and evidence for an interdomain carboxy-terminal helix in ornithine transcarbamoylase.

Predictions of tertiary structures of proteins from their amino acid sequences are facilitated greatly when the structures of homologous proteins are known. On this basis, structural features of Escherichia coli ornithine transcarbamoylase (OTCase) were investigated by site-directed mutagenesis experiments based on the known tertiary structure of the catalytic (c) chain of E. coli aspartate transcarbamoylase (ATCase). In ATCase, each c chain is composed of two globular domains connected by two interdomain helices, one of which is near the C-terminus and is critical for the in vivo folding of the chains and their assembly into trimers. Each active site is located at the interface between two chains and requires the participation of residues from each of the adjacent chains. OTCase, a trimeric enzyme, has been proposed to be similar in structure to the ATCase trimer on the basis of sequence identity (32%), the nature of the reaction catalyzed by the enzyme, and secondary structure predictions. As shown here, analysis of OTCase and ATCase sequences revealed extensive evolutionary conservation in portions corresponding to the ATCase active site and the C-terminal helix. Truncations and substitutions within the predicted C-terminal helix of OTCase had effects on activity and thermal stability strikingly similar to those caused by analogous alterations in ATCase. Similarly, substitutions at either of two conserved residues, Ser 55 and Lys 86, in the proposed active site of OTCase had deleterious effects parallel to those caused by the analogous ATCase substitutions. Hybrid trimers comprised of chains from both these relatively inactive OTCase mutants exhibited dramatically increased activity, as predicted for shared active sites located at the chain interfaces. These results strongly support the hypothesis that the tertiary and quaternary structures of the two enzymes are similar.     

Use of L-asparagine and N-phosphonacetyl-L-asparagine to investigate the linkage of catalysis and homotropic cooperativity in E. coli aspartate transcarbomoylase

The mechanism of domain closure and the allosteric transition of Escherichia coli aspartate transcarbamoylase (ATCase) are investigated using L-Asn, in the presence of carbamoyl phosphate (CP), and N-phosphonacetyl-L-asparagine (PASN). ATCase was found to catalyze the carbamoylation of L-Asn with a K(m) of 122 mM and a maximal velocity 10-fold lower than observed with the natural substrate, L-Asp. As opposed to L-Asp, no cooperativity was observed with respect to L-Asn. Time-resolved small-angle X-ray scattering (SAXS) and fluorescence experiments revealed that the combination of CP and L-Asn did not convert the enzyme from the T to the R state. PASN was found to be a potent inhibitor of ATCase exhibiting a K(D) of 8.8 microM. SAXS experiments showed that PASN was able to convert the entire population of molecules to the R state. Analysis of the crystal structure of the enzyme in the presence of PASN revealed that the binding of PASN was similar to that of the R-state complex of ATCase with N-phosphonaceyl-L-aspartate, another potent inhibitor of the enzyme. The linking of CP and L-Asn into one molecule, PASN, correctly orients the asparagine moiety in the active site to induce domain closure and the allosteric transition. This entropic effect allows for the high affinity binding of PASN. However, the binding of L-Asn, in the presence of a saturating concentration of CP, does not induce the closure of the two domains of the catalytic chain, nor does the enzyme undergo the transition to the high-activity high- affinity R structure. These results imply that Arg229, which interacts with the beta-carboxylate of L-Asp, plays a critical role in the orientation of L-Asp in the active site and demonstrates the requirement of the beta-carboxylate of L-Asp in the mechanism of domain closure and the allosteric transition in E. coli ATCase.     

Allosteric control of quaternary states in E. coli aspartate transcarbamylase

Changes in the molecular dimensions of ATCase in the unligated T-state are an increase of 0.4 A in the separation of catalytic trimers when ATP binds. When the R-state is produced by binding of phosphonoacetamide and malonate, addition of CTP or CTP + UTP decreases the separation of catalytic trimers by 0.5 A. In the unliganded Glu239----Gln mutant, in which the T-state is destabilized so that the enzyme exists in an intermediate quaternary state, ligation of ATP transforms the mutant enzyme to the R-state, whereas CTP converts this enzyme to the T-state. Thus, this mutant is much more sensitive to heterotropic allosteric control than is the native enzyme. In this communication we propose a preliminary model based on new crystallographic results that heterotropic regulation occurs partly through control of the quaternary structure by these effectors, thus regulating catalysis.     

The allosteric activator Mg-ATP modifies the quaternary structure of the R-state of Escherichia coli aspartate transcarbamylase without altering the T<-->R equilibrium

The allosteric enzyme aspartate transcarbamylase from Escherichia coli (ATCase) displays regulatory properties that involve various conformational changes, including a large quaternary structure rearrangement. This entails a major change in its solution X-ray scattering curve upon binding substrate analogues. We show here that, in the presence of the nucleotide effector ATP, known to stimulate the enzyme activity, the scattering profiles show a marked dependence on the metal bound to ATP. Whereas ATP has no major effect on the scattering pattern of ATCase, a saturating concentration of Mg-ATP notably modifies the scattering profile of the enzyme, either in the absence or in the presence of the bisubstrate analogue N-(phosphonacetyl)-l-aspartate (PALA). The transition with PALA in the presence of this metal-nucleotide complex remains concerted. Furthermore, Mg-ATP, as already observed with ATP, has no detectable direct effect on the T to R transition. The experimental scattering curves in the presence of Mg-ATP were fitted by a modeling approach using rigid body movements of the regulatory subunits and the catalytic trimers in the crystal structures. While the differences observed in the T-state in the presence of Mg-ATP are essentially attributed to the binding per se of the nucleotide, the solution structure of the R-state complexed to Mg-ATP is even more extended along the 3-fold axis than the previously described R solution structure, which is already more stretched out along the same axis than the crystal R structure. Based on the crystal structure of the enzyme in the R-state complexed with free ATP, a proposal is made to account for the effect of magnesium.     

Comparative modeling of mammalian aspartate transcarbamylase

Mammalian aspartate transcarbamylase (ATCase) is part of a 243 kDa multidomain polypeptide, called CAD, that catalyzes the first three steps in de novo pyrimidine biosynthesis. The structural organization of the mammalian enzyme is very different from E. coli ATCase, a dodecameric, monofunctional molecule comprised of six copies of separate catalytic and regulatory chains. Nevertheless, sequence similarities and other properties suggested that the mammalian ATCase domain and the E. coli ATCase catalytic chain have the same tertiary fold. A model of mammalian ATCase was built using the X-ray coordinates of the E. coli catalytic chain as a tertiary template. Five small insertions and deletions could be readily accommodated in the model structure. Following energy minimization the RMS difference in the alpha carbon positions of the mammalian and bacterial proteins was 0.93 A. A comparison of the hydrophobic energies, surface accessibility index, and the distribution of hydrophilic and hydrophobic residues of the CAD ATCase structure with correctly and incorrectly folded proteins and with several X-ray structures supported the validity of the model. The mammalian ATCase domain associates to form a compact globular trimer, a prerequisite for catalysis since the active site is comprised of residues from adjacent subunits. Interactions between the clearly defined aspartate and carbamyl phosphate subdomains of the monomer were largely preserved while there was appreciable remodeling of the trimeric interfaces. Several clusters of basic residues are located on the upper surface of the domain which account in part for the elevated isoelectric point (pI = 9.4) and may represent contact regions with other more acidic domains within the chimeric polypeptide. A long interdomain linker connects the monomer at its upper surface to the remainder of the polypeptide. The configuration of active site residues is virtually identical in the mammalian and bacterial enzymes. While the CAD ATCase domain can undergo the local conformational changes that accompany catalysis in the E. coli enzyme, the high activity, closed conformation is probably more stable in the mammalian enzyme.     

Negative complementation in aspartate transcarbamylase. Analysis of hybrid enzyme molecules containing different arrangements of polypeptide chains from wild-type and inactive mutant catalytic subunits.

A comprehensive set of hybrid molecules of aspartate transcarbamylase (ATCase) from Escherichia coli has been constructed of wild-type and mutationally altered catalytic chains. The mutant enzymes that were virtually devoid of activity contained a replacement of Gly-128 in the catalytic polypeptide chains by either Asp or Arg. The kinetic properties of these hybrid enzyme-like molecules were analyzed to evaluate the basis for the unusual quaternary constraint demonstrated by an intersubunit hybrid containing one wild-type catalytic subunit, one inactive mutant subunit (containing the Gly to Asp replacement), and three wild-type regulatory subunits. A similar intersubunit hybrid was constructed from the wild-type catalytic subunit and the mutant in which Gly-128 was replaced by Arg, and it too demonstrated a pronounced decrease in activity relative to that expected for a hybrid containing three active sites. Moreover, neither of these hybrid holoenzymes exhibited the cooperativity with respect to aspartate that is characteristic of wild-type ATCase. In contrast, hybrid holoenzymes containing at least one wild-type chain in each catalytic subunit showed cooperativity. Also, hybrid enzymes containing different arrangements of five, four, three, or two wild-type catalytic chains with an appropriate complement of mutant chains had specific activities proportional to the number of wild-type chains in the holoenzymes. Exceptions were observed only in hybrids in which one of the two subunits in the holoenzyme was composed completely of mutant catalytic chains. For these hybrids the negative complementation was manifested as a much lower enzyme activity than expected from the number of wild-type chains in the enzyme and the loss of cooperativity. Thus, the activity and allosteric properties of these hybrids is dependent on the arrangement of catalytic chains in the holoenzyme, in contrast to results obtained for hybrids containing native and chemically modified catalytic chains. Intrasubunit hybrid catalytic trimers containing one or two wild-type chains exhibited one-third and two-thirds the activity of the intact wild-type catalytic subunit, respectively, indicating the dominant negative effect that was seen in intersubunit hybrid holoenzymes is absent within trimers.     

Importance of a conserved residue, aspartate-162, for the function of Escherichia coli aspartate transcarbamoylase.

Aspartate-162 in the catalytic chain of aspartate transcarbamoylase is conserved in all of the sequences determined to date. The X-ray structure of the Escherichia coli enzyme indicates that this residue is located in a loop region (160's loop) that is near the interface between two catalytic trimers and is also close to the active site. In order to test whether this conserved residue is important for support of the internal architecture of the enzyme and/or involved in transmitting homotropic and heterotropic effects, the function of this residue was studied using a mutant version of the enzyme with an alanine at this position (Asp-162----Ala) created by site-specific mutagenesis. The Asp-162----Ala enzyme exhibits a 400-fold reduction in the maximal observed specific activity, approximately 2-fold and 10-fold decreases in the aspartate and carbamoyl phosphate concentrations at half the maximal observed specific activity respectively, a loss of homotropic cooperativity, and loss of response to the regulatory nucleotides ATP and CTP. Furthermore, equilibrium binding studies indicate that the affinity of the mutant enzyme for CTP is reduced more than 10-fold. The isolated catalytic subunit exhibits a 660-fold reduction in maximal observed specific activity compared to the wild-type catalytic subunit. The Km values for aspartate and carbamoyl phosphate for the Asp-162----Ala catalytic subunit were within 2-fold of the values observed for the wild-type catalytic subunit. Computer simulations of the energy-minimized mutant enzyme indicate that the space once occupied by the side chain of Asp-162 may be filled by other side chains, suggesting that Asp-162 is important for stabilizing the internal architecture of the wild-type enzyme.     

Site-specific substitutions of the Tyr-165 residue in the catalytic chain of aspartate transcarbamoylase promotes a T-state preference in the holoenzyme

The amino acid residue Tyr-165C of aspartate transcarbamoylase (EC 2.1.3.2) of Escherichia coli has been proposed to be involved in the transition from the T-state to the R-state upon binding of the bisubstrate analogue N-(phosphonacetyl)-L-aspartate. Site-specific mutagenesis has been used to substitute phenylalanine for tyrosine, thus maintaining the aromatic R-group but removing the charged hydroxyl moiety. This mutation dramatically altered the aspartate requirements for the holoenzyme but did not substantially affect the homotropic or heterotropic characteristics of the oligomer. The aspartate requirements for half-maximal saturation increased from 5.5 mM at pH 7.0 for the native holoenzyme to approximately 90 mM in the mutant enzyme. Nonetheless, estimates of the kinetic cooperativity index remained similar (Hill coefficients: Tyr-165C, n = 2.1; Phe-165C, n = 2.5). CTP inhibited both enzymes approximately 70% and ATP activated approximately 40% at the aspartate concentrations required for half-maximal saturation (5 and 90 mM, respectively). The maximal velocity of the mutant holoenzyme is almost identical to that of the wild-type enzyme. The phenylalanine substitution does not affect the stability of the holoenzyme to heat or mercurials, and the Vmax of the catalytic trimer was 444% greater than that of the holoenzyme. Upon dissociation of the wild-type native enzyme into catalytic trimers, the Vmax increased 450%. The Km for aspartate in the separated catalytic trimer is approximately 2-fold higher than for the native catalytic trimer (16.5 versus 8 mM at pH 7.0). It is clear from the data that although Tyr-165C is not directly involved in the active site of the enzyme, it does play a pivotal role in catalytic transitions of the holoenzyme. In addition, the homotropic and heterotropic characteristics of the enzyme do not seem to be altered by the substitution of phenylalanine for Tyr-165C in the E. coli aspartate transcarbamoylase, although other substitutions have been reported (Robey, E. H., and Schachman, H. K. (1984) J. Biol. Chem. 259, 11180-11183) which show more complex effects.     

Structural model of the R state of Escherichia coli aspartate transcarbamoylase with substrates bound

The allosteric enzyme aspartate transcarbamoylase (ATCase) exists in two conformational states. The enzyme, in the absence of substrates is primarily in the low-activity T state, is converted to the high-activity R state upon substrate binding, and remains in the R state until substrates are exhausted. These conformational changes have made it difficult to obtain structural data on R-state active-site complexes. Here we report the R-state structure of ATCase with the substrate Asp and the substrate analog phosphonoactamide (PAM) bound. This R-state structure represents the stage in the catalytic mechanism immediately before the formation of the covalent bond between the nitrogen of the amino group of Asp and the carbonyl carbon of carbamoyl phosphate. The binding mode of the PAM is similar to the binding mode of the phosphonate moiety of N-(phosphonoacetyl)-l-aspartate (PALA), the carboxylates of Asp interact with the same residues that interact with the carboxylates of PALA, although the position and orientations are shifted. The amino group of Asp is 2.9 A away from the carbonyl oxygen of PAM, positioned correctly for the nucleophilic attack. Arg105 and Leu267 in the catalytic chain interact with PAM and Asp and help to position the substrates correctly for catalysis. This structure fills a key gap in the structural determination of each of the steps in the catalytic cycle. By combining these data with previously determined structures we can now visualize the allosteric transition through detailed atomic motions that underlie the molecular mechanism.     

Intersubunit hydrogen bond acts as a global molecular switch in Escherichia coli aspartate transcarbamoylase

Tyr 165 in the catalytic subunit of Escherichia coli aspartate transcarbamoylase (ATCase, EC 2.1.3.2) forms an intersubunit hydrogen bond in the T state with Glu 239 in the 240s loop of a second catalytic subunit, which is broken in the T to R transition. Substitution of Tyr 165 by Phe lowers substrate affinity by approximately an order of magnitude and alters the pH profile for enzyme function. We have determined the crystal structure of Y165F at 2.4 Å resolution by molecular replacement, using a wild-type T state structure as the probe, and refined it to an R value of 25.2%. The Y165F mutation induces a global conformational change that is in the opposite direction to the T to R transition and therefore results in an extreme T state. The two catalytic trimers move closer by ∼0.14 Å and rotate by ∼0.2°, in the opposite direction to the T→R rotation; the two domains of each catalytic chain rotate by ∼2.1°, also in the opposite direction to the T→R transition; and the 240s loop adopts a new conformation. Residues 229 to 236 shift by ∼2.4 Å so that the active site is more open. Residues 237 to 244 rotate by ∼24.1°, altering interactions within the 240s loop and at the C1-C4 and C1-R4 interfaces. Arg 167, a key residue in domain closure and interactions with L-Asp, swings out from the active site to interact with Tyr 197. This crystal structure is consistent with the functional properties of Y165F, expands our knowledge of the conformational repertoire of ATCase, and indicates that the canonical T state does not represent an extreme. Proteins 33:430–443, 1998. © 1998 Wiley-Liss, Inc.     

Crystallographic snapshots of the complete catalytic cycle of the unregulated aspartate transcarbamoylase from Bacillus subtilis

Here, we report high-resolution X-ray structures of Bacillus subtilis aspartate transcarbamoylase (ATCase), an enzyme that catalyzes one of the first reactions in pyrimidine nucleotide biosynthesis. Structures of the enzyme have been determined in the absence of ligands, in the presence of the substrate carbamoyl phosphate, and in the presence of the bisubstrate/transition state analog N-phosphonacetyl-l-aspartate. Combining the structural data with in silico docking and electrostatic calculations, we have been able to visualize each step in the catalytic cycle of ATCase, from the ordered binding of the substrates, to the formation and decomposition of the tetrahedral intermediate, to the ordered release of the products from the active site. Analysis of the conformational changes associated with these steps provides a rationale for the lack of cooperativity in trimeric ATCases that do not possess regulatory subunits.     

A cis-proline to alanine mutant of E. coli aspartate transcarbamoylase: kinetic studies and three-dimensional crystal structures

The only cis-proline residue in Escherichia coli aspartate transcarbamoylase has been replaced by alanine using site-specific mutagenesis. The Pro268-->Ala enzyme exhibits a 40-fold reduction in enzyme activity and decreased substrate affinity toward carbamoyl phosphate and aspartate compared to the corresponding values for the wild-type enzyme. The concentration of the bisubstrate analogue N-phosphonacetyl-L-aspartate (PALA) required to activate the mutant enzyme to the same extent as the wild-type enzyme is significantly increased. The heterotropic effects of ATP and CTP upon the Pro268-->Ala enzyme are also altered. Crystal structures of the Pro268-->Ala enzyme in both T- and R-states show that the cis-peptidyl linkage between Leu267 and Ala268 is maintained. However, the tertiary structure of both the catalytic and regulatory chains has been altered by the amino acid substitution, and the mobility of the active-site residues is increased for the R-state structure of Pro268-->Ala enzyme as comparison with the wild-type R-state structure. These structural changes are responsible for the loss of enzyme activity. Thus, Pro268 is required for the proper positioning of catalytically critical residues in the active site and is important for the formation of the high-activity high-affinity R-state of E. coli aspartate transcarbamoylase.     

Modelling allosteric processes in E coli aspartate transcarbamylase

The allosteric properties of aspartate transcarbamylase from E coli have been investigated by a combination of genetic, biochemical and structural studies. Based on the X-ray structures of the enzyme in T and R state established by Lipscomb et al, we have analyzed the interactions between the 12 polypeptide chains and have identified subunit interfaces that play a major part in the allosteric mechanism: the c1c4 interface between the 2 catalytic trimers, and one of 2 different interfaces between catalytic and regulatory chains, the c1r4 interface, which exists only in T state. We have modelled mutations affecting these interfaces: mutation pAR5 in the gene coding for r chains concerns the c1r4 interface, mutation Tyr----Phe 240 in the gene coding for c chains, the c1c4 interface. Both mutant proteins have reduced cooperativity and/or allosteric regulation by CTP and ATP. Molecular mechanic simulations lead to specific proposals for the structural origin of these effects, and some of the proposals can be checked by site-directed mutagenesis. Finally, we have modelled substrates bound at the active site of the T state, which binds aspartate less tightly than the R state and for which X-ray structures of bound substrate analogs were not available.     

Reconstitution of active catalytic trimer of aspartate transcarbamoylase from proteolytically cleaved polypeptide chains.

Treatment of the catalytic (C) trimer of Escherichia coli aspartate transcarbamoylase (ATCase) with alpha-chymotrypsin by a procedure similar to that used by Chan and Enns (1978, Can. J. Biochem. 56, 654-658) has been shown to yield an intact, active, proteolytically cleaved trimer containing polypeptide fragments of 26,000 and 8,000 MW. Vmax of the proteolytically cleaved trimer (CPC) is 75% that of the wild-type C trimer, whereas Km for aspartate and Kd for the bisubstrate analog, N-(phosphonacetyl)-L-aspartate, are increased about 7- and 15-fold, respectively. CPC trimer is very stable to heat denaturation as shown by differential scanning microcalorimetry. Amino-terminal sequence analyses as well as results from electrospray ionization mass spectrometry indicate that the limited chymotryptic digestion involves the rupture of only a single peptide bond leading to the production of two fragments corresponding to residues 1-240 and 241-310. This cleavage site involving the bond between Tyr 240 and Ala 241 is in a surface loop known to be involved in intersubunit contacts between the upper and lower C trimers in ATCase when it is in the T conformation. Reconstituted holoenzyme comprising two CPC trimers and three wild-type regulatory (R) dimers was shown by enzyme assays to be devoid of the homotropic and heterotropic allosteric properties characteristic of wild-type ATCase. Moreover, sedimentation velocity experiments demonstrate that the holoenzyme reconstituted from CPC trimers is in the R conformation. These results indicate that the intact flexible loop containing Tyr 240 is essential for stabilizing the T conformation of ATCase. Following denaturation of the CPC trimer in 4.7 M urea and dilution of the solution, the separate proteolytic fragments re-associate to form active trimers in about 60% yield. How this refolding of the fragments, docking, and association to form trimers are achieved is not known.     

Crystal structure of T state aspartate carbamoyltransferase of the hyperthermophilic archaeon Sulfolobus acidocaldarius

Aspartate carbamoyltransferase (ATCase) is a model enzyme for understanding allosteric effects. The dodecameric complex exists in two main states (T and R) that differ substantially in their quaternary structure and their affinity for various ligands. Many hypotheses have resulted from the structure of the Escherichia coli ATCase, but so far other crystal structures to test these have been lacking. Here, we present the tertiary and quaternary structure of the T state ATCase of the hyperthermophilic archaeon Sulfolobus acidocaldarius (SaATC(T)), determined by X-ray crystallography to 2.6A resolution. The quaternary structure differs from the E.coli ATCase, by having altered interfaces between the catalytic (C) and regulatory (R) subunits, and the presence of a novel C1-R2 type interface. Conformational differences in the 240 s loop region of the C chain and the C-terminal region of the R chain affect intersubunit and interdomain interfaces implicated previously in the allosteric behavior of E.coli ATCase. The allosteric-zinc binding domain interface is strengthened at the expense of a weakened R1-C4 type interface. The increased hydrophobicity of the C1-R1 type interface may stabilize the quaternary structure. Catalytic trimers of the S.acidocaldarius ATCase are unstable due to a drastic weakening of the C1-C2 interface. The hyperthermophilic ATCase presents an interesting example of how an allosteric enzyme can adapt to higher temperatures. The structural rearrangement of this thermophilic ATCase may well promote its thermal stability at the expense of changes in the allosteric behavior.     

T-state active site of aspartate transcarbamylase: crystal structure of the carbamyl phosphate and L-alanosine ligated enzyme

An X-ray diffraction study to 2.0 A resolution shows that this enzyme, ATCase, is in the T-state (the c3 to c3 distance is 45.2 A) when ATCase is bound to carbamyl phosphate (CP) and to L-alanosine (an analogue of aspartate). This result strongly supports the kinetic results that alanosine did not inhibit the carbamylation of aspartate in the normal reaction of native ATCase plus CP and aspartate [Baillon, J., Tauc, P., and Hervé, G. (1985) Biochemistry 24, 7182-7187]. The structure further reveals that the phosphate of CP is 4 A away from its known position in the R-state and is in the T-state position of P(i) in a recent study of ATCase complexed with products, phosphate (P(i)) and N-carbamyl-L-aspartate [Huang, J., and Lipscomb, W. N. (2004) Biochemistry 43, 6422-6426]. Moreover, the alanosine position in this T-state is somewhat displaced from that expected for its analogue, aspartate, from the R-state position. The relations of these structural aspects to the kinetics are presented.     

Cooperative binding of the bisubstrate analog N-(phosphonacetyl)-L-aspartate to aspartate transcarbamoylase and the heterotropic effects of ATP and CTP

Most investigations of the allosteric properties of the regulatory enzyme aspartate transcarbamoylase (ATCase) from Escherichia coli are based on the sigmoidal dependence of enzyme activity on substrate concentration and the effects of the inhibitor, CTP, and the activator, ATP, on the saturation curves. Interpretations of these effects in terms of molecular models are complicated by the inability to distinguish between changes in substrate binding and catalytic turnover accompanying the allosteric transition. In an effort to eliminate this ambiguity, the binding of the 3H-labeled bisubstrate analog N-(phosphonacetyl)-L-aspartate (PALA) to aspartate transcarbamoylase in the absence and presence of the allosteric effectors ATP and CTP has been measured directly by equilibrium dialysis at pH 7 in phosphate buffer. PALA binds with marked cooperativity to the holoenzyme with an average dissociation constant of 110 nM. ATP and CTP alter both the average affinity of ATCase for PALA and the degree of cooperativity in the binding process in a manner analogous to their effects on the kinetic properties of the enzyme; the average dissociation constant of PALA decreases to 65 nM in the presence of ATP and increases to 266 nM in the presence of CTP while the Hill coefficient, which is 1.95 in the absence of effectors, becomes 1.35 and 2.27 in the presence of ATP and CTP, respectively. The isolated catalytic subunit of ATCase, which lacks the cooperative kinetic properties of the holoenzyme, exhibits only a very slight degree of cooperativity in binding PALA. The dissociation constant of PALA from the catalytic subunit is 95 nM. Interpretation of these results in terms of a thermodynamic scheme linking PALA binding to the assembly of ATCase from catalytic and regulatory subunits demonstrates that saturation of the enzyme with PALA shifts the equilibrium between holoenzyme and subunits slightly toward dissociation. Ligation of the regulatory subunits by either of the allosteric effectors leads to a change in the effect of PALA on the association-dissociation equilibrium.     

In vivo formation of allosteric aspartate transcarbamoylase containing circularly permuted catalytic polypeptide chains: implications for protein folding and assembly.

Because the N- and C-terminal amino acids of the catalytic (c) polypeptide chains of Escherichia coli aspartate transcarbamoylase (ATCase) are in close proximity to each other, it has been possible to form in vivo five different active ATCase variants in which the terminal regions of the wild-type c chains are linked in a continuous polypeptide chain and new termini are introduced elsewhere in either of the two structural domains of the c chain. These circularly permuted (cp) chains were produced by constructing tandem pyrB genes, which encode the c chain of ATCase, followed by application of PCR. Chains expressed in this way assemble efficiently in vivo to form active, stable ATCase variants. Three such variants have been purified and shown to have the kinetic and physical properties characteristic of wild-type ATCase composed of two catalytic (C) trimers and three regulatory (R) dimers. The values of Vmax for cpATCase122, cpATCase222, and cpATCase281 ranged from 16-21 mumol carbamoylaspartate per microgram per h, compared with 15 for wild-type ATCase, and the values for K0.5 for the variants were 4-17 mM aspartate, whereas wild-type ATCase exhibited a value of 6 mM. Hill coefficients for the three variants varied from 1.8 to 2.1, compared with 1.4 for the wild-type enzyme. As observed with wild-type ATCase, ATP activated the variants containing the circularly permuted chains, as shown by the lowering of K0.5 for aspartate and a decrease in the Hill coefficient (nH). In contrast, CTP caused both an increase in K0.5 and nH for the variants, just as observed with wild-type ATCase. Thus, the enzyme containing the permuted chains with widely diverse N- and C-termini exhibited the homotropic and heterotropic effects characteristic of wild-type ATCase. The decrease in the sedimentation coefficient of the variants caused by the binding of the bisubstrate ligand N-(phosphonacetyl)-L-aspartate (PALA) was also virtually identical to that obtained with wild-type ATCase, thereby indicating that these altered ATCase molecules undergo the analogous ligand-promoted allosteric transition from the taut (T) state to the relaxed (R) conformation. These ATCase molecules with new N- and C-termini widely dispersed throughout the c chains are valuable models for studying in vivo and in vitro folding of polypeptide chains.     

T-state inhibitors of E. coli aspartate transcarbamoylase that prevent the allosteric transition

Escherichia coli aspartate transcarbamoylase (ATCase) catalyzes the committed step in pyrimidine nucleotide biosynthesis, the reaction between carbamoyl phosphate (CP) and l-aspartate to form N-carbamoyl-l-aspartate and inorganic phosphate. The enzyme exhibits homotropic cooperativity and is allosterically regulated. Upon binding l-aspartate in the presence of a saturating concentration of CP, the enzyme is converted from the low-activity low-affinity T state to the high-activity high-affinity R state. The potent inhibitor N-phosphonacetyl-l-aspartate (PALA), which combines the binding features of Asp and CP into one molecule, has been shown to induce the allosteric transition to the R state. In the presence of only CP, the enzyme is the T structure with the active site primed for the binding of aspartate. In a structure of the enzyme-CP complex (T(CP)), two CP molecules were observed in the active site approximately 7A apart, one with high occupancy and one with low occupancy. The high occupancy site corresponds to the position for CP observed in the structure of the enzyme with CP and the aspartate analogue succinate bound. The position of the second CP is in a unique site and does not overlap with the aspartate binding site. As a means to generate a new class of inhibitors for ATCase, the domain-open T state of the enzyme was targeted. We designed, synthesized, and characterized three inhibitors that were composed of two phosphonacetamide groups linked together. These two phosphonacetamide groups mimic the positions of the two CP molecules in the T(CP) structure. X-ray crystal structures of ATCase-inhibitor complexes revealed that each of these inhibitors bind to the T state of the enzyme and occupy the active site area. As opposed to the binding of Asp in the presence of CP or PALA, these inhibitors are unable to initiate the global T to R conformational change. Although the best of these T-state inhibitors only has a K(i) value in the micromolar range, the structural information with respect to their mode of binding provides important information for the design of second generation inhibitors that will have even higher affinity for the active site of the T state of the enzyme.