An aspartate transcarbamylase lacking catalytic subunit interactions. Study of conformational changes by ultraviolet absorbance and circular dichroism spectroscopy.

A modified form of aspartate transcarbamylase is synthesized by Escherichia coli in the presence of 2-thiouracil which does not exhibit homotropic cooperative interactions between active sites yet retains heterotropic cooperative interactions due to nucleotide binding. The conformational changes induced in the modified enzyme by the binding of different ligands (substrates, substrate analogs, a transition state analog, and nucleotide effectors) were studied using ultraviolet absorbance and circular dichroism difference spectroscopy. Comparison of the results for the modified enzyme and its isolated subunits to those for the native enzyme and its isolated subunits showed that the conformational changes detected by these methods are qualitatively similar in the two enzymes. Comparison of the absorbance difference spectra due to the binding of a transition substrate analog to the intact native or modified enzymes to the corresponding results for the isolated subunits suggested that ligand binding causes an increased exposure to solvent of certain tyrosyl and phenylalanyl residues in the intact enzymes but not in the isolated subunits. This result is consistent with a diminution of subunit contacts due to substrate binding in the course of homotropic interactions in the native enzyme. Such conformational changes, though perhaps necessary for homotropic cooperativity, are not sufficient to cause homotropic cooperativity since the modified enzyme gave identical perturbations. Interactions of the transition state analog, N-(phosphonacetyl)-L-aspartate, with the modified enzyme were studied. Enzyme kinetic data obtained at low aspartate concentrations showed that this transition state analog does not stimulate activity, but rather exhibits the inhibition predicted for the total absence of homotropic cooperative interactions in the modified enzyme. Spectrophotometric titrations of the number of catalytic sites with the transition state analog showed that the modified enzyme and its isolated subunits possess, respectively, four and two high affinity sites for the inhibitor instead of six and three observed in the case of the normal enzyme and its isolated catalytic subunits. These results are correlated with the lower specific enzymatic activities of the modified enzyme and its catalytic subunits compared to the normal corresponding enzymatic species.     

The stimulation of Escherichia coli aspartate transcarbamylase activity by adenosine triphosphate. Relation with the other regulatory conformational changes; a model.

The process of stimulation of Escherichia coli aspartate transcarbamylase activity by ATP was investigated. The efficiency of the phenomenon increases with the number of phosphate groups bound to adenosine. The pH dependence of the stimulation by ATP and adenylyl methylenediphosphonate indicates that the binding of these nucleotides requires the ionization of their last phosphate acidic group. The aspartate trans-carbamylase activity does not appear to be under the influence of the “energy charge ratio” but rather to depend directly on the ATP concentration. The stimulation decreases when the aspartate concentration increases. Saturating amounts of ATP do not provoke the shift in optimum pH for the catalytic activity which is shown to be associated with the homotropic co-operative interactions between the catalytic sites. This result provides additional evidence that homotropic and heterotropic interactions correspond to different molecular mechanisms. ATP reverses the effect of the feedback inhibitor CTP. A model is proposed to account for the relationship between the process of stimulation by ATP and the other regulatory conformational changes.     

L-alanosine: a noncooperative substrate for Escherichia coli aspartate transcarbamylase

L-Alanosine, an antibiotic produced by Streptomyces alanosinicus, can be used by Escherichia coli aspartate transcarbamylase as a substrate instead of L-aspartate. The Michaelis constant of the catalytic subunit for this analogue is about 10 times higher than that for the physiological substrate, and the catalytic constant is about 30 times lower. The saturation curve of the native enzyme for L-alanosine indicates the lack of homotropic cooperative interactions between the catalytic sites for the utilization of this compound. It appears therefore that L-alanosine is unable to promote the allosteric transition. However, N-(phosphonoacetyl)-L-aspartate, a "bisubstrate analogue" of the physiological substrates, stimulates the reaction. This phenomenon is very similar to that reported by Foote and Lipscomb [Foote, J., & Lipscomb, W. N. (1981) J. Biol. Chem. 256, 11428-11433] concerning the reverse reaction using carbamylaspartate. The reaction is normally sensitive to the physiological effectors ATP and CTP. The significance of these results for the mechanism of the allosteric regulation is discussed.     

An aspartate transcarbamylase lacking catalytic subunit interactions. II. Regulatory subunits are responsible for the lack of co-operative interactions between catalytic sites. Drastic feedback inhibition does not restore these interactions

It has been previously reported (Kerbiriou &; Hervé, 1972) that, when a uracil-requiring mutant of Escherichia coli is derepressed for the biosynthesis of the enzymes of the pyrimidine pathway in the presence of 2-thiouracil, it synthesizes a modified aspartate transcarbamylase which is still sensitive to the feedback inhibitor CTP, but which does not show homotropic positive interactions between catalytic sites. It is shown here that these homotropic interactions do not reappear upon strong inhibition by CTP, indicating that the two types of interactions are really disconnected and must involve different molecular mechanisms. CTP is acting at the level of the apparent K_m of the enzyme for aspartate. It is also the case for ATP, which stimulates 2-thiouracil aspartate-transcarbamylase. Kinetic studies of the hybrid molecules made up of subunits prepared from normal and modified enzymes show that it is a modification at the level of the regulatory subunits which is responsible for the lack of co-operative interactions between catalytic sites. These results are discussed in terms of a four-state model.     

Plant aspartate transcarbamylase: an affinity chromatographic method for the purification of the enzyme from germinated seedings.

A simple and rapid affinity chromatographic method for the isolation of aspartate transcarbamylase from germinated seedlings of mung bean (Phaseolus aureus) was developed. A partially purified preparation of the enzyme was chromatographed on an affinity column containing aspartate linked to CNBr-activated Sepharose 4B. Aspartate transcarbamylase was specifically eluted from the column with 10 mm aspartate or 0.5 m KCl. The enzyme migrated as a single sharp band during disc electrophoresis at pH 8.6 on polyacrylamide gels. Electrophoresis of the sodium dodecyl sulfate-treated enzyme showed two distinct protein bands, suggesting that the mung bean aspartate transcarbamylase was made up of nonidentical subunits. Like the enzyme purified by conventional procedures, this enzyme preparation also exhibited positive homotropic interactions with carbamyl phosphate and negative heterotropic interactions with UMP. This method was extended to the purification of aspartate transcarbamylase from Lathyrus sativus, Eleucine coracona, and Trigonella foenum graecum.     

A possible model for the concerted allosteric transition in Escherichia coli aspartate transcarbamylase as deduced from site-directed mutagenesis studies

Aspartate transcarbamylase is stabilized in a low-affinity-low-activity state exhibiting no cooperativity by selective perturbation of the Glu-50-Arg-167 and Glu-50-Arg-234 interdomain salt bridges. Similarly, a high-affinity-high-activity state of the enzyme, retaining a significant amount of cooperativity, is obtained by perturbation of the interaction between Tyr-240 and Asp-271. In this work, we show that the rupture of the link between Tyr-240 and Asp-271 in the enzyme already lacking the interdomain salt bridges regenerates the homotropic cooperative interactions between the catalytic sites and substantially increases the activity and affinity of the enzyme for aspartate. These results suggest a possible relationship between these two sets of interactions for the establishment of the cooperative behavior of the enzyme. Another mutation, Glu-239 to Gln, introduced to perturb the Glu-239-Lys-164 and Glu-239-Tyr-165 interactions between the two catalytic subunits, is sufficient to "lock" the enzyme in the R state. These observations emphasize the importance of the interactions at the interface between the catalytic trimers in maintaining the T state of the enzyme and shed light on the role played by this pathway in the communication of homotropic cooperativity between the different sites. A model including all these findings, as well as the interactions stabilizing the T state or the R state in the presence of the natural substrates, is proposed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulatory Properties of Intergeneric Hybrids of Aspartate Transcarbamylase

THE regulatory enzyme aspartate transcarbamylase (ATCase) from Escherichia coli contains two non-identical protein sub-units, one the catalytic subunit which provides the active sites of the enzyme and the other the regulatory subunit which provides the binding sites for nucleotide inhibitors and activators^1,2. The catalytic subunit is a trimer of “C” polypeptide chains, associated by three heterologous c: c domains of bonding (terminology given by Monod et al.³ and Cohlberg et al.⁴). The regulatory subunit is a dimer of “R” chains, associated by an isologous r: r domain. Two catalytic and three regulatory subunits interact specifically across six r: c domains of inter-subunit bonding to complete the quaternary structure of the ATCase molecule.     

Cooperativity and high pressure: stabilization of the R conformation of the allosteric aspartate transcarbamylase under the influence of pressure.

The allosteric enzyme aspartate transcarbamylase (ATCase) from E. coli shows homotropic cooperative interactions between its six catalytic sites for the binding of the substrate aspartate. This cooperativity is explained by the transition of the enzyme from a conformation which has a low affinity for aspartate (T state) to a conformation with high affinity (R state). The crystallographic structures of these two conformations are known to a resolution of 2.5 A and 2.1 A, respectively, and they reveal an important difference in the quaternary structure of the protein. Enzyme kinetics under high pressure were used to study the transition between the two states. It appears that in the presence of a low concentration of aspartate, conditions under which the enzyme is essentially in the T state, pressure promotes the transition to the R state, the maximal effect being observed at 120 MPa. This transition is accompagnied by a significant deltaV. This observation is in accordance with the change in the protein surface exposed to the solvent, and with the increased number of water molecules bound to the protein. Since the partial specific volume of the enzyme does not change significantly during the T to R transition, the negative deltaV is only related to the change in hydration of the protein. This result emphasizes a significant role of the protein-solvent interactions in this important regulatory conformational change.     

Co-operative interactions between the catalytic sites in Escherichia coli aspartate transcarbamylase. Role of the C-terminal region of the regulatory chains

In aspartate transcarbamylase (ATCase) each regulatory chain interacts with two catalytic chains each one belonging to a different trimeric catalytic subunit (R1-C1 and R1-C4 types of interactions as defined in Fig. 1). In order to investigate the interchain contacts that are involved in the co-operative interactions between the catalytic sites, a series of modified forms of the enzyme was prepared by site-directed mutagenesis. The amino acid replacements were devised on the basis of the previously described properties of an altered form of ATCase (pAR5-ATCase) which lacks the homotropic co-operative interactions between the catalytic sites. The results obtained (enzyme kinetics, bisubstrate analog influence and pH studies) show that the R1-C4 interaction is essential for the establishment of the enzyme conformation that has a low affinity for aspartate (T state), and consequently for the existence of co-operativity between the catalytic sites. This interaction involves the 236-250 region of the aspartate binding domain of the catalytic chain (240s loop) and the 143-149 region of the regulatory chain which comprises helix H3'.     

Structure-function relationship in allosteric aspartate carbamoyltransferase from Escherichia coli. II. Involvement of the C-terminal region of the regulatory chain in homotropic and heterotropic interactions

The modified aspartate transcarbamylase (ATCase) encoded by the transducing phage described by Cunin et al. has been purified to homogeneity. In this altered form of enzyme (pAR5-ATCase) the last eight amino acids of the C-terminal end of the regulatory chains are replaced by a sequence of six amino acids coded for by the lambda DNA. This modification has very informative consequences on the allosteric properties of ATCase. pAR5-ATCase lacks the homotropic co-operative interactions between the catalytic sites for aspartate binding and is "frozen" in the R state. In addition, this altered form of enzyme is insensitive to the physiological feedback inhibitor CTP, in spite of the fact that this nucleotide binds normally to the regulatory sites. Conversely, pAR5-ATCase is fully sensitive to the activator ATP. However, this activation is limited to the extent of the previously described "primary effect" as expected from an ATCase form "frozen" in the R state. These results emphasize the importance of the three-dimensional structure of the C-terminal region of the regulatory chains for both homotropic and heterotropic interactions. In addition, they indicate that the primary effects of CTP and ATP involve different features of the regulatory chain-catalytic chain interaction area.     

Intramolecular signal transmission in enterobacterial aspartate transcarbamylases II. Engineering co-operativity and allosteric regulation in the aspartate transcarbamylase of Erwinia herbicola

The aspartate transcarbamylase (ATCase) from Erwinia herbicola differs from the other investigated enterobacterial ATCases by its absence of homotropic co-operativity toward the substrate aspartate and its lack of response to ATP which is an allosteric effector (activator) of this family of enzymes. Nevertheless, the E. herbicola ATCase has the same quaternary structure, two trimers of catalytic chains with three dimers of regulatory chains ((c3)2(r2)3), as other enterobacterial ATCases and shows extensive primary structure conservation. In (c3)2(r2)3 ATCases, the association of the catalytic subunits c3 with the regulatory subunits r2 is responsible for the establishment of positive co-operativity between catalytic sites for the binding of aspartate and it dictates the pattern of allosteric response toward nucleotide effectors. Alignment of the primary sequence of the regulatory polypeptides from the E. herbicola and from the paradigmatic Escherichia coli ATCases reveals major blocks of divergence, corresponding to discrete structural elements in the E. coli enzyme. Chimeric ATCases were constructed by exchanging these blocks of divergent sequence between these two ATCases. It was found that the amino acid composition of the outermost beta-strand of a five-stranded beta-sheet in the effector-binding domain of the regulatory polypeptide is responsible for the lack of co-operativity and response to ATP of the E. herbicola ATCase. A novel structural element involved in allosteric signal recognition and transmission in this family of ATCases was thus identified.     

A loop involving catalytic chain residues 230-245 is essential for the stabilization of both allosteric forms of Escherichia coli aspartate transcarbamylase

The allosteric transition of Escherichia coli aspartate transcarbamylase involves significant alterations in structure at both the quaternary and tertiary levels. On the tertiary level, the 240s loop (residues 230-245 of the catalytic chain) repositions, influencing the conformation of Arg-229, a residue near the aspartate binding site. In the T state, Arg-229 is bent out of the active site and may be stabilized in this position by an interaction with Glu-272. In the R state, the conformation of Arg-229 changes, allowing it to interact with the beta-carboxylate of aspartate, and is stabilized in this position by a specific interaction with Glu-233. In order to ascertain the function of Arg-229, Glu-233, and Glu-272 in the catalytic and cooperative interactions of the enzyme, three mutant enzymes were created by site-specific mutagenesis. Arg-229 was replaced by Ala, while both Glu-233 and Glu-272 were replaced by Ser. The Arg-229----Ala and Glu-233----Ser enzymes exhibit 10,000-fold and 80-fold decreases in maximal activity, respectively, and they both exhibit a 2-fold increase in the aspartate concentration at half the maximal observed velocity, [S]0.5. The Arg-229----Ala enzyme still exhibits substantial homotropic cooperativity, but all cooperativity is lost in the Glu-233----Ser enzyme. The Glu-233----Ser enzyme also shows a 4-fold decrease in the carbamyl phosphate [S]0.5, while the Arg-229----Ala enzyme shows no change in the carbamyl phosphate [S]0.5 compared to the wild-type enzyme. The Glu-272 to Ser mutation results in a slight reduction in maximal activity, an increase in [S]0.5 for both aspartate and carbamyl phosphate, and reduced cooperativity. Analysis of the isolated catalytic subunits from these three mutant enzymes reveals that in each case the changes in the kinetic properties of the isolated catalytic subunit are similar to the changes caused by the mutation in the holoenzyme. PALA was able to activate the Glu-233----Ser enzyme, at low aspartate concentrations, even though the mutant holoenzyme did not exhibit any cooperativity, indicating that cooperative interactions still exist between the active sites in this enzyme. It is proposed that Glu-233 of the 240s loop helps create the high-activity-high-affinity R state by positioning the side chain of Arg-229 for aspartate binding while Glu-272 helps stabilize the low-activity-low-affinity T state by positioning the side chain of Arg-229 so that it cannot interact with aspartate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Function of arginine-234 and aspartic acid-271 in domain closure, cooperativity, and catalysis in Escherichia coli aspartate transcarbamylase

Two mutant versions of Escherichia coli aspartate transcarbamylase were created by site-specific mutagenesis. Arg-234 of the 240s loop was replaced by serine in order to help deduce the function of the interactions that normally occur between Arg-234 and both Glu-50 and Gln-231 in the R state of the enzyme. The other mutation involved the replacement of Asp-271 by asparagine to further test the functional importance of the Tyr-240-Asp-271 link that has previously been proposed to stabilize the T state of the enzyme [Middleton, S. A., & Kantrowitz, E. R. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 5866-5870]. The Arg-234----Ser holoenzyme exhibits no cooperativity, a 24-fold reduction in maximal velocity, normal affinity for carbamyl phosphate, and substantially reduced affinity for aspartate and N-(phosphonoacetyl)-L-aspartate (PALA). Unlike the wild-type enzyme, the heterotropic effectors ATP and CTP are able to influence the activity of the Arg-234----Ser enzyme at saturating aspartate concentrations. The Arg-234----Ser catalytic subunit exhibits a 33-fold reduction in maximal activity, an aspartate Km of 261 mM, compared to 5.7 mM for the wild-type catalytic subunit, and only a small alteration in the Km for carbamyl phosphate. Together these results provide additional evidence that the interdomain bridging interactions between Glu-50 of the carbamyl phosphate domain and both Arg-167 and Arg-234 of the aspartate domain are necessary for the stabilization of the high-activity-high-affinity configuration of the active site of the enzyme. Furthermore, without the interdomain bridging interactions, the holoenzyme no longer exhibits homotropic cooperativity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Discrimination between nucleotide effector responses of aspartate transcarbamoylase due to a single site substitution in the allosteric binding site

The substitution of alanine for lysine at position 56 of the regulatory polypeptide of aspartate transcarbamoylase affected both homotropic and heterotropic characteristics. In the absence of effectors, the ALAr56-substituted holoenzyme lost the homotropic cooperativity observed for aspartate in the wild-type holoenzyme. Under conditions of allosteric inhibition in the presence of 2mM CTP, the cooperative character of ATCase was restored, and the Hill coefficient increased from 1.0 to 1.7. In contrast to the native enzyme, the altered enzyme did not respond to ATP; however, ATP could still bind to the enzyme as demonstrated by its direct competition with CTP. Furthermore, the recently observed CTP-UTP synergism of the wild-type enzyme was not detectable. The site-directed mutant enzyme could not be activated by low levels of the bisubstrate analogue, N-(phosphonacetyl)-L-aspartate, and the rate of association of pHMB with the cysteine residues located at the interface of the catalytic and regulatory chains was slightly altered. These characteristics suggested that the mutant holoenzyme assumed a relaxed (or abnormal T state) conformation. Thus, this single substitution differentially affected the heterotropic responses to the various allosteric effectors of ATCase and eliminated the homotropic characteristics in response to aspartate in the absence of CTP.     

Functionally important arginine residues of aspartate transcarbamylase.

The reaction of phenylglyoxal with aspartate transcarbamylase and its isolated catalytic subunit results in complete loss of enzymatic activity (Kantrowitz, E. R., and Lipscomb, W. N. (1976) J. Biol. Chem. 251, 2688-2695). If N-(phosphonacetyl)-L-aspartate is used to protect the active site, we find that phenylglyoxal causes destruction of the enzyme's susceptibility to activation by ATP and inhibition by CTP. Furthermore, CTP only minimally protects the regulatory site from reaction with this reagent. The modified enzyme still binds CTP although with reduced affinity. After reaction with phenylglyoxal, the native enzyme shows reduced cooperativity. The hybrid with modified regulatory subunits and native catalytic subunits exhibits slight heterotropic or homotropic properties, while the reverse hybrid, with modified catalytic subunits and native regulatory subunits, shows much reduced homotropic properties but practically normal heterotropic interactions. The decrease in the ability of CTP to inhibit the enzyme correlates with the loss of 2 arginine residues/regulatory chain (Mr = 17,000). Under these reaction conditions, 1 arginine residue is also modified on each catalytic chain (Mr = 33,000). Reaction rate studies of p-hydroxymercuribenzoate, with the liganded and unliganded modified enzyme suggest that the reaction with phenylglyoxal locks the enzyme into the liganded conformation. The conformational state of the regulatory subunit is implicated as having a critical role in the expression of the enzyme's heterotropic and homotropic properties.     

On the detection of homotropic effects in enzymes of low co-operativity. Application to modified aspartate transcarbamoylase

In contrast with the ease of observing heterotropic effects in allosteric enzymes of low co-operativity, the detection of homotropic effects is often difficult. As a consequence, erroneous conclusions about the uncoupling of homotropic and heterotropic effects can result unless sensitive techniques are used for analyzing the kinetic data. Simulations of experiments as well as actual measurements on the allosteric enzyme, aspartate transcarbamoylase, of Escherichia coli and some of its modified forms, were performed in attempts to develop stringent diagnostic procedures for the detection of homotropic effects in enzymes of low co-operativity. The analyses show that direct saturation plots (velocity versus substrate concentration), double reciprocal plots, and Hill plots yield misleading results in that the co-operativity known to be present is not observed. In contrast, Eadie plots (velocity/substrate concentration versus velocity) are much more sensitive in revealing homotropic effects. Since the observed co-operativity depends on both the allosteric equilibrium constant, L, and the number of active sites, n, simulations were performed on the effect of those parameters. The maxima in the Eadie plots increased as L was lowered and conversely the maxima decreased as n was reduced. These changes were confirmed with a mutant aspartate transcarbamoylase which had the same specific activity as the wild-type enzyme and a lower value of L, and also with a hybrid enzyme containing fewer active sites and the same L value. Analogous experiments on nitrated aspartate transcarbamoylase derivatives of decreasing activity showed that Eadie plots were of value in distinguishing between the changes in L and n values resulting from the inactivation. Data from the literature were analyzed in the form of Eadie plots and in all cases homotropic effects were readily detectable for aspartate transcarbamoylase derivatives previously claimed to be devoid of co-operativity.     

Crystal and molecular structures of native and CTP-liganded aspartate carbamoyltransferase from Escherichia coli

Atomic models representing the electron density of two crystalline forms of aspartate carbamoyltransferase from Escherichia coli are reported here. The unliganded form (R32 crystal symmetry) and the CTP-liganded form (P321 crystal symmetry) have been refined independently at resolutions of 3.0 å and 2.8 Å, respectively, each to a crystallographic R-factor of 27%. The molecular models include at least 95% of the theoretical number of atoms for the aspartate Carbamoyltransferase molecule based on chemical sequence information. We provide details of the refinement process for the two structures, and an evaluation of the accuracy of the molecular models.For the most part, the regulatory and catalytic chains of the unliganded enzyme and the CTP-liganded form are in similar conformations. Large conformational differences in the CTP and native forms exist, however, specifically in the region of CTP binding to the regulatory chain. In addition, a segment of ten amino acid residues, which includes Lys83 and Lys84 of the catalytic chain, is disordered in the CTP-liganded form, in contrast to the native structure, where the same residues have refined well into density.Each catalytic monomer of aspartate carbamoyltransferase is in contact with three catalytic chains and two regulatory monomers. Each regulatory monomer borders on one other regulatory chain and two catalytic chains. The catalytic trimera are in contact in the hexamer; residues important to homotropic effects and catalysis (Tyr165 and Tyr232) are integral parts of the interface. We present a thorough survey of interface regions, cataloging polar interactions between sidechains throughout the molecule.We discuss, in context with the present structures, the chemical modifications and mutations of the enzyme. Highlighted specifically are Cys47, Tyr165 and Tyr232, Lys83, Lys84, Trp209 and Trp279 and Gly128, residues of demonstrated importance to the catalytic of regulatory function or aspartate carbamoyltransferase. The spatial arrangement of “active site” residues argues for a catalytic pocket shared between two monomers within catalytic subunit.     

Regulation of the pyruvate kinase from Alcaligenes eutrophus H 16 in vitro and in vivo

The biosynthesis of the enzyme pyruvate kinase (E.C. 2.7.1.40) of Alcaligenes eutrophus (Hydrogenomonas eutropha) H 16 was influenced by the carbon and energy source. After growth on gluconate the specific enzyme activity was high while acetate grown cells exhibited lower activities (340 and 55 μmoles/min·g protein, respectively). The pyruvate kinase from autotrophically grown cells was purified 110-fold. The enzyme was characterized by homotropic cooperative interactions with the substrate phosphoenolpyruvate, the activators AMP, ribose-5-phosphate, glucose-6-phosphate and the inhibitor ortho-phosphate. In addition to phosphate ATP caused inhibition but in this case non-sigmoidal kinetics was obtained. The half maximal substrate saturation constant S_0.5 for phosphoenolpyruvate in the absence of any effectors was 0.12 mM, in the presence of 1 mM ribose-5-phosphate 0.07 mM, and with 9 mM phosphate 0.67 mM. The corresponding Hill values were 0.96, 1.1 and 2.75. The ADP saturation curve was hyperbolic even in the presence of the effectors, the K _m value was 0.14 mM ADP. When the known intracellular metabolite concentrations in A. eutrophus H 16 were compared with the regulatory sensitivity of the enzyme, it appeared that under the conditions in vivo the inhibition by ATP was more important than the regulation by the allosteric effectors.     

The contribution of individual interchain interactions to the stabilization of the T and R states of Escherichia coli aspartate transcarbamoylase

Stabilization of the T and R allosteric states of Escherichia coli aspartate transcarbamoylase is governed by specific intra- and interchain interactions. The six interchain interactions between Glu-239 in one catalytic chain of one catalytic trimer with both Lys-164 and Tyr-165 of a different catalytic chain in the other catalytic trimer have been shown to be involved in the stabilization of the T state. In this study a series of hybrid versions of aspartate transcarbamoylase was studied to determine the minimum number of these Glu-239 interactions necessary to maintain homotropic cooperativity and the T allosteric state. Hybrids with zero, one, and two Glu-239 stabilizing interactions do not exhibit cooperativity, whereas the hybrids with three or more Glu-239 stabilizing interactions exhibit cooperativity. The hybrid enzymes with one or more of the Glu-239 stabilizing interactions also exhibit heterotropic interactions. Two hybrids with three Glu-239 stabilizing interactions, in different geometric relationships, had identical properties. From this and previous studies, it is concluded that the 239 stabilizing interactions play a critical role in the manifestation of homotropic cooperativity in aspartate transcarbamoylase by the stabilization of the T state of the enzyme. As substrate binding energy is utilized, more and more of the T state stabilizing interactions are relaxed, and finally the enzyme shifts to the R state. In the case of the Glu-239 stabilizing interactions more than three of the interactions must be broken before the enzyme shifts to the R state. The interactions between the catalytic and regulatory chains and between the two catalytic trimers of aspartate transcarbamoylase provide a global set of interlocking interactions that stabilize the T and R states of the enzyme. The substrate-induced local conformational changes observed in the structure of the isolated catalytic subunit drive the quaternary T to R transition of aspartate transcarbamoylase and functionally induced homotropic cooperativity.