Amino acid substitutions which stabilize aspartate transcarbamoylase in the R state disrupt both homotropic and heterotropic effects

We have used site-specific amino acid substitutions to investigate the linkage between the allosteric properties of arpartate transcarbamoylase and the global conformational transition exhibited by the enzyme upon binding active-site ligands. Two mutationally altered enzymes in which an amino acid substitution had been introduced at a single position in the catalytic polypeptide chain (Lys-164----Glu and Glu-239----Lys) and a third species harboring both of these substitutions (Lys-164:Glu-239----Glu:Lys) were constructed. Sedimentation velocity difference studies were performed in order to assess the effects of the amino acid substitutions on the quaternary structure of the holoenzyme in the absence and presence of various active-site ligands, including the bisubstrate analog, N-(phosphonacetyl)-L-aspartate (PALA), which has been shown previously to promote the allosteric transition. In the absence of ligand, two of the mutationally altered enzymes, Lys-164----Glu and Lys-164:Glu-239----Glu:Lys, existed in the R conformation, isomorphous with that of the PALA-liganded wild-type holoenzyme. These enzymes exhibited no conformational change upon binding PALA. The unliganded Glu-239----Lys enzyme had an average sedimentation coefficient intermediate between that of the unliganded and PALA-liganded states of the wild-type enzyme which could be accounted for in terms of a mixture of T- and R-state molecules. This mutant enzyme was converted to the fully swollen conformation upon binding PALA, phosphate or carbamoyl phosphate. The allosteric properties of the mutationally altered species were investigated by PALA-binding studies and by steady-state enzyme kinetics. In each case, the mutationally altered enzymes were devoid of both homotropic and heterotropic effects, supporting the premise that the allosteric properties of the wild-type enzyme are linked to a ligand-promoted change in quaternary structure.     

The 80s loop of the catalytic chain of Escherichia coli aspartate transcarbamoylase is critical for catalysis and homotropic cooperativity.

The X-ray structure of the Escherichia coli aspartate transcarbamoylase with the bisubstrate analog phosphonacetyl-L-aspartate (PALA) bound shows that PALA interacts with Lys84 from an adjacent catalytic chain. To probe the function of Lys84, site-specific mutagenesis was used to convert Lys84 to alanine, threonine, and asparagine. The K84N and K84T enzymes exhibited 0.08 and 0.29% of the activity of the wild-type enzyme, respectively. However, the K84A enzyme retained 12% of the activity of the wild-type enzyme. For each of these enzymes, the affinity for aspartate was reduced 5- to 10-fold, and the affinity for carbamoyl phosphate was reduced 10- to 30-fold. The enzymes K84N and K84T exhibited no appreciable cooperativity, whereas the K84A enzyme exhibited a Hill coefficient of 1.8. The residual cooperativity and enhanced activity of the K84A enzyme suggest that in this enzyme another mechanism functions to restore catalytic activity. Modeling studies as well as molecular dynamics simulations suggest that in the case of only the K84A enzyme, the lysine residue at position 83 can reorient into the active site and complement for the loss of Lys84. This hypothesis was tested by the creation and analysis of the K83A enzyme and a double mutant enzyme (DM) that has both Lys83 and Lys84 replaced by alanine. The DM enzyme has no cooperativity and exhibited 0.18% of wild-type activity, while the K83A enzyme exhibited 61% of wild-type activity. These data suggest that Lys84 is not only catalytically important, but is also essential for binding both substrates and creation of the high-activity, high-affinity active site. Since low-angle X-ray scattering demonstrated that the mutant enzymes can be converted to the R-structural state, the loss of cooperativity must be related to the inability of these mutant enzymes to form the high-activity, high-affinity active site characteristic of the R-functional state of the enzyme.     

On the mechanism of assembly of the aspartate transcarbamoylase from Escherichia coli.

The mechanism of subunit assembly of aspartate transcarbamoylase from Escherichia coli was studied by following the kinetics of reassociation. The isolated trimetric catalytic subunit (c3) and dimeric regulatory subunit (r2) were mixed together and formation of the dodecameric native enzyme (c6r6) was monitored by measuring changes in activity. Under appropriate conditions the reassociation was second order with respect to the c3 concentration and the effects of varying r2 concentration on the second-order rate constant were examined. An optimum R2 concentration of about 0.07 micrometer was observed. A scheme of the assembly pathways is proposed and is based on the reversible formation of c3r2n (n = 0, 1, 2 or 3) as intermediates. Various combinations of two such c3r2n species are considered as possible rate-limiting steps. This model yields an expression which relates the experimentally determined (overall) second-order rate constant to the equilibrium constant (Kd) governing the formation of c3r2n, the r2 concentration, and four coefficients which reflect the contribution of different types of assembly processes. Using previously determined values of Kd, the above expression for each r2 concentration reduces to a linear equation with four unknowns. The experimental data were subjected to multiple linear-regression analysis and values for the four coefficients were found which gave an excellent fit. Our results show that reassociation of the subunits is a fast bimolecular reaction with rate constants in excess of 10(6) M-1 s-1. Our analysis also suggests that interactions involving a total of more than three r2 subunits (e.g. the combination of c3r2 with c3r6) might contribute significantly to the overall assembly. The influence of various ligands on the reassociation rate profile was also studied. All ligands examined were partially inhibitory to the formation of native enzyme. The effects of substrates were similar to those of CTP whereas the effects of ATP were substantially different. These observations can be readily interpreted by postulating different conformational changes induced by the ligands. These changes should alter the relative orientation of the subunit contacts which must be formed in the reassociation process. The interpretation is consistent with our previous model of the allosteric mechanism.     

The contribution of threonine 55 to catalysis in aspartate transcarbamoylase.

Heavy-atom isotope effects and steady-state kinetic parameters were measured for the catalytic trimer of an active site mutant of aspartate transcarbamoylase, T55A, to assess the role of Thr 55 in catalysis. The binding of carbamoyl phosphate to the T55A mutant was decreased by 2 orders of magnitude relative to the wild-type enzyme whereas the affinities for aspartate and succinate were not markedly altered. This indicates that Thr 55 plays a significant role in the binding of CbmP. If, as had been suggested previously, Thr 55 assists in the polarization of the carbonyl group of CbmP, the carbon isotope effect for the T55A mutant should increase relative to that observed for the wild-type enzyme. However, the opposite is seen, indicating that Thr 55 is not involved in stabilizing the oxyanion in the transition state. Quantitative analysis of a series of 13C and 15N isotope effects suggested that the rate-determining step in the reaction catalyzed by T55A trimer may be a conformational change in the protein subsequent to formation of the Michaelis complex. Thus, Thr 55 may facilitate a conformational change in the enzyme that is a prerequisite for catalysis. An altered active site environment in the binary and Michaelis complexes with T55A trimer is reflected in the pH profiles for log V, log (V/K)asp, and pK(i) succinate, show a displacement in the pK values of ionizing residues involved in aspartate binding and catalysis relative to the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inactivation of wheat-germ aspartate transcarbamoylase by the arginine-specific reagent phenylglyoxal.

Wheat-germ aspartate transcarbamoylase (EC 2.1.3.2) was inactivated by phenylglyoxal in a first-order process, provided that the inactivation time did not exceed 10 min. Apparent first-order rate constants were linearly dependent on phenylglyoxal concentration, indicating a bimolecular reaction between a single active-centre residue and phenylglyoxal, with second-order constant of 0.023 mM-1 X min-1. A plot of apparent first-order rate constant versus pH showed a steep rise above pH 9.5, indicating that the essential residue has a pKa value of 10.5 or higher, consistent with an arginine residue. Saturating concentrations of the following ligands provided a degree of protection (percentages in parentheses) against 1 mM-phenylglyoxal: N-phosphonoacetyl-L-aspartate, a bisubstrate analogue (94%); carbamoyl phosphate (75%); UMP, an end-product inhibitor (53%). Succinate (an analogue of L-aspartate) alone gave no protection, but in combination with carbamoyl phosphate raised the protection to 92%, in agreement with the known binding order of the two substrates. These results indicate that the essential arginine residue is close to the carbamoyl phosphate site, probably oriented towards the aspartate site. Attempts to desensitize the UMP-binding site by reaction with phenylglyoxal, while protecting the active centre, were unsuccessful. The essential active-centre arginine residue is compared with a similar residue in the Escherichia coli enzyme.     

Characterization of the aspartate transcarbamoylase from Methanococcus jannaschii

The genes from the thermophilic archaeabacterium Methanococcus jannaschii that code for the putative catalytic and regulatory chains of aspartate transcarbamoylase were expressed at high levels in Escherichia coli. Only the M. jannaschii PyrB (Mj-PyrB) gene product exhibited catalytic activity. A purification protocol was devised for the Mj-PyrB and M. jannaschii PyrI (Mj-PyrI) gene products. Molecular weight measurements of the Mj-PyrB and Mj-PyrI gene products revealed that the Mj-PyrB gene product is a trimer and the Mj-PyrI gene product is a dimer. Preliminary characterization of the aspartate transcarbamoylase from M. jannaschii cell-free extract revealed that the enzyme has a similar molecular weight to that of the E. coli holoenzyme. Kinetic analysis of the M. jannaschii aspartate transcarbamoylase from the cell-free extract indicates that the enzyme exhibited limited homotropic cooperativity and little if any regulatory properties. The purified Mj-catalytic trimer exhibited hyperbolic kinetics, with an activation energy similar to that observed for the E. coli catalytic trimer. Homology models of the Mj-PyrB and Mj-PyrI gene products were constructed based on the three-dimensional structures of the homologous E. coli proteins. The residues known to be critical for catalysis, regulation, and formation of the quaternary structure from the well characterized E. coli aspartate transcarbamoylase were compared.     

Three of the six possible intersubunit stabilizing interactions involving Glu-239 are sufficient for restoration of the homotropic and heterotropic properties of Escherichia coli aspartate transcarbamoylase

A hybrid version of Escherichia coli aspartate transcarbamoylase was investigated in which one catalytic subunit has the wild-type sequence, and the other catalytic subunit has Glu-239 replaced by Gln. Since Glu-239 is involved in intersubunit interactions, this hybrid could be used to evaluate the extent to which T state stabilization is required for homotropic cooperativity and for heterotropic effects. Reconstitution of the hybrid holoenzyme (two different catalytic subunits with three wild-type regulatory subunits) was followed by separation of the mixture by anion-exchange chromatography. To make possible the resolution of the three holoenzyme species formed by the reconstitution, the charge of one of the catalytic subunits was altered by the addition of six aspartic acid residues to the C terminus of each of the catalytic chains (AT-C catalytic subunit). Control experiments indicated that the AT-C catalytic subunit as well as the holoenzyme formed with AT-C and wild-type regulatory subunits had essentially the same homotropic and heterotropic properties as the native catalytic subunit and holoenzyme, indicating that the addition of the aspartate tail did not influence the function of either enzyme. The control reconstituted holoenzyme, in which both catalytic subunits have Glu-239 replaced by Gln, exhibited no cooperativity, an enhanced affinity for aspartate, and essentially no heterotropic response identical to the enzyme isolated without reconstitution. The hybrid containing one normal and one mutant catalytic subunit exhibited homotropic cooperativity with a Hill coefficient of 1.4 and responded to the nucleotide effectors at about 50% of the level of the wild-type enzyme. Small angle x-ray scattering experiments with the hybrid enzyme indicated that in the absence of ligands it was structurally similar, but not identical, to the T state of the wild-type enzyme. In contrast to the wild-type enzyme, addition of carbamoyl phosphate induced a significant alteration in the scattering pattern, whereas the bisubstrate analog N-phosphonoacetyl-L-aspartate induced a significant change in the scattering pattern indicating the transition to the R-structural state. These data indicate that in the hybrid enzyme only three of the usual six interchain interactions involving Glu-239 are sufficient to stabilize the enzyme in a low affinity, low activity state and allow an allosteric transition to occur.     

Regulatory kinetics of wheat-germ aspartate transcarbamoylase. Adaptation of the concerted model to account for complex kinetic effects of uridine 5''-monophosphate.

The kinetic effects of the end-product inhibitor UMP on aspartate transcarbamoylase (EC 2.1.3.2) purified to homogeneity from wheat germ were studied. In agreement with an earlier study of the relatively crude enzyme [Yon (1972) Biochem. J. 128, 311-320], the half-saturating concentrations of UMP and of the first substrate, carbamoyl phosphate (but not of the second, L-aspartate), were found to be strongly interdependent. However, the kinetic behaviour of the pure enzyme differed from that of the crude enzyme in several important respects, namely: (a) the apparent affinity for UMP was lower with the pure enzyme; (b) sigmoidicity was absent from plots of initial rate versus carbamoyl phosphate concentration, each at a fixed UMP concentration; (c) sigmoidicity was greatly exaggerated in plots of initial rate versus UMP concentration, each at a fixed carbamoyl phosphate concentration, owing to the occurrence of a slight but definite maximum in each plot at low UMP concentration; (d) there was a relative increase in this maximum in the presence of N-phosphonacetyl-L-aspartate, an inhibitor competitive with carbamoyl phosphate. It is shown that a modified two-conformation concerted-transition model can be used to account for most of these features of the pure enzyme. The model treats carbamoyl phosphate and UMP as antagonistic allosteric ligands binding to alternative conformational states [Monod, Wyman & Changeux (1965) J. Mol. Biol. 12, 88-118], carbamoyl phosphate binding non-exclusively (dissociation constants 20 microM and 85 microM respectively) and UMP binding exclusively (dissociation constant 2.5 microM). The model postulates further that the conformation with lower affinity for carbamoyl phosphate has the higher value of kcat., and that it binds UMP in competition with carbamoyl phosphate. Parameters giving the best fit of experimental data to this model were found by a non-linear least-squares search procedure.     

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.     

Primary structure and properties of an inactive mutant aspartate transcarbamoylase.

A mutant form of Escherichia coli aspartate transcarbamoylase (ATCase) which lacks catalytic activity has been purified and characterized (Wall, K.A., Flatgaard, J.E., Gibbons, I., and Schachman, H.K. (1979) J. Biol. Chem 254, 11910-11916). Peptide mapping of the mutant and wild type catalytic chains followed by the determination of the amino acid sequence of the one altered peptide in the mutant indicated that a glycyl residue was replaced by aspartic acid. This substitution is located at position 125 in the tentative sequence kindly provided by W. Konigsberg (personal communication). The mutant protein has an overall secondary structure similar to that of the wild type as indicated by circular dichroism spectroscopy. However, marked changes in the reactivity of several amino acid residues were demonstrated. Lysyl residue 84 which in the wild type subunits reacts specifically with pyridoxal 5'-phosphate is only slightly reactive in the mutant even though the peptide containing that residue was not altered in amino acid composition. Another residue, cysteinyl 46, which is thought to be in the active site, is much more reactive toward p-hydroxymercuribenzoate in the mutant subunit than in the wild type protein. Finally, tyrosyl residue 213, which according to recent crystallographic studies is not near the active site and which exhibits an unusually low pK (9.1) in the wild type catalytic subunits, appears to have its pK shifted to 10.5 or higher as a result of the mutation. The evidence indicates that the substitution of an aspartyl for a glycyl residue at a region of the amino acid sequence remote from those residues in the active site causes sufficient modification of the tertiary structure to cause the loss of enzyme activity and to affect the reactivity of other residues in the protein. Moreover, the quaternary structure of the intact enzyme is altered as well since the subunit interactions are greatly weakened.     

Ionization of amino acid residues involved in the catalytic mechanism of aspartate transcarbamoylase.

The chemical and kinetic mechanisms of the reaction catalyzed by the catalytic trimer of aspartate transcarbamoylase have been examined. The variation of the kinetic parameters with pH indicated that at least four ionizing amino acid residues are involved in substrate binding and catalysis. The pH dependence of K(ia) for carbamoyl phosphate and the K(i) for N-(phosphonoacetyl)-L- aspartate revealed that a protonated residue with a pK value of 9.0 is required for the binding of carbamoyl phosphate. However, the variation with pH of K(i) for succinate, a competitive inhibitor of aspartate, and for cysteine sulfinate, a slow substrate, showed that a single residue with a pK value of 7.3 must be protonated for binding these analogues and, by inference, aspartate. The profile of log V against pH displayed a decrease in reaction rate at low and high pH, suggesting that two groups associated with the Michaelis complex, a deprotonated residue with a pK value of 7.2 and a protonated group with a pK value of 9.5, are involved in catalysis. By contrast, the catalytically productive form of the enzyme-carbamoyl phosphate complex, as illustrated in the bell-shaped pH dependence of log (V/K)(asp), is one in which a residue with a pK value of 7.0 must be protonated while a group with a pK value of 9.1 is deprotonated. This interpretation is supported by the results from the temperature dependence of the V and V/K profiles and from the pH dependence of pK(i) for the aspartate analogues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and in vitro evaluation of aspartate transcarbamoylase inhibitors

The design, synthesis, and evaluation of a series of novel inhibitors of aspartate transcarbamoylase (ATCase) are reported. Several submicromolar phosphorus-containing inhibitors are described, but all-carboxylate compounds are inactive. Compounds were synthesized to probe the postulated cyclic transition-state of the enzyme-catalyzed reaction. In addition, the associated role of the protonation state at the phosphorus acid moiety was evaluated using phosphinic and carboxylic acids. Although none of the synthesized inhibitors is more potent than N-phosphonacetyl-l-aspartate (PALA), the compounds provide useful mechanistic information, as well as the basis for the design of future inhibitors and/or prodrugs.     

Subunit coupling and kinetic co-operativity of polymeric enzymes. Amplification, attenuation and inversion effects

The principles of structural kinetics, as applied to dimeric enzymes, allow us to understand how the strength of subunit coupling controls both substrate-binding co-operativity, under equilibrium conditions, and kinetic co-operativity, under steady state conditions. When subunits are loosely coupled, positive substrate-binding co-operativity may result in either an inhibition by excess substrate or a positive kinetic co-operativity. Alternatively, negative substrate-binding co-operativity is of necessity accompanied by negative kinetic co-operativity. Whereas the extent of negative kinetic co-operativity is attenuated with respect to the corresponding substrate-binding co-operativity, the positive kinetic co-operativity is amplified with respect to that of the substrate-binding co-operativity. Strong kinetic co-operativity cannot be generated by a loose coupling of subunits. If subunit is propagated to the other, the dimeric enzyme may display apparently surprising co-operativity effects. If the strain of the active sites generated by subunit coupling is relieved in the non-liganded and fully-liganded states, both substrate-binding co-operativity and kinetic co-operativity cannot be negative. If the strain of the active sites however, is not relieved in these states, negative substrate-binding co-operativity is accompanied by either a positive or a negative co-operativity. The possible occurrence of a reversal of kinetic co-operativity, with respect to substrate-binding co-operativity, is the direct consequence of quaternary constraints in the dimeric enzyme. Moreover, tight coupling between subunits may generate a positive kinetic co-operativity which is not associated with any substrate-binding co-operativity. In other words a dimeric enzyme may well bind the substrate in a non co-operative fashion and display a positive kinetic co-operativity generated by the strain of the active sites.     

Active-site-directed inactivation of wheat-germ aspartate transcarbamoylase by pyridoxal 5''-phosphate.

Treatment of 1 microM wheat-germ aspartate transcarbamoylase with 1 mM-pyridoxal 5'-phosphate caused a rapid loss of activity, concomitant with the formation of a Schiff base. Complete loss of activity occurred within 10 min when the Schiff base was reduced with a 100-fold excess of NaBH4. Concomitantly, one amino group per chain was modified. No further residues were modified in the ensuing 30 min. The kinetics of inactivation were examined under conditions where the Schiff base was reduced before assay. Inactivation was apparently first-order. The pseudo-first-order rate constant, kapp., showed a hyperbolic dependence upon the concentration of pyridoxal 5'-phosphate, suggesting that the enzyme first formed a non-covalent complex with the reagent, modification of a lysine then proceeding within this complex. Inactivation of the enzyme by pyridoxal was 20 times slower than that by pyridoxal 5'-phosphate, indicating that the phosphate group was important in forming the initial complex. Partial protection against pyridoxal phosphate was provided by the leading substrate, carbamoyl phosphate, and nearly complete protection was provided by the bisubstrate analogue, N-phosphonoacetyl-L-aspartate, and the ligand-pair carbamoyl phosphate plus succinate. Steady-state kinetic studies, under conditions that minimized inactivation, showed that pyridoxal 5'-phosphate was also a competitive inhibitor with respect to the leading substrate, carbamoyl phosphate. Pyridoxal 5'-phosphate therefore appears to be an active-site-directed reagent. A sample of the enzyme containing one reduced pyridoxyl group per chain was digested with trypsin, and the labelled peptide was isolated and shown to contain a single pyridoxyl-lysine residue. Partial sequencing around the labelled lysine showed little homology with the sequence surrounding lysine-84, an active-centre residue of the catalytic subunit of aspartate transcarbamoylase from Escherichia coli, whose reaction with pyridoxal 5'-phosphate shows many similarities to the results described in the present paper. Arguably the reactive lysine is conserved between the two enzymes whereas the residues immediately surrounding the lysine are not. The same conclusion has been drawn in a comparison of reactive histidine residues in the two enzymes [Cole & Yon (1986) Biochemistry 25, 7168-7174].     

Ligand-mediated conformational changes in wheat-germ aspartate transcarbamoylase indicated by proteolytic susceptibility.

Ligand-mediated effects on the inactivation of pure wheat-germ aspartate transcarbamoylase by trypsin were examined. Inactivation was apparently first-order in all cases, and the effects of ligand concentration on the pseudo-first-order rate constant, k, were studied. Increase in k (labilization) was effected by carbamoyl phosphate, phosphate and the putative transition-state analogue, N-phosphonoacetyl-L-aspartate. Decrease in k (protection) was effected by the end-product inhibitor, UMP, and by the ligand pairs aspartate/phosphate and succinate/carbamoyl phosphate, but not by aspartate or succinate alone up to 10 mM. Except for protection by the latter ligand pairs, all other ligand-mediated effects were also observed on inactivation of the enzyme by Pronase and chymotrypsin. Ligand-mediated effects on the fragmentation of the polypeptide chain by trypsin were examined electrophoretically. Slight labilization of the chain was observed in the presence of carbamoyl phosphate, phosphate and N-phosphonoacetyl-L-aspartate. An extensive protection by UMP was observed, which apparently included all trypsin-sensitive peptide bonds. No significant effect by the ligand pair succinate/carbamoyl phosphate was noted. It is concluded from these observations that UMP triggers an extensive, probably co-operative, transition to a proteinase-resistant conformation, and that carbamoyl phosphate similarly triggers a transition to an alternative, proteinase-sensitive, conformation. These antagonistic conformational changes may account for the regulatory kinetic effects reported elsewhere [Yon (1984) Biochem. J. 221, 281-287]. The protective effect by the ligand pairs aspartate/phosphate and succinate/carbamoyl phosphate, which operates only against trypsin, is concluded to be due to local shielding of essential lysine or arginine residues in the aspartate-binding pocket of the active site, to which aspartate (or its analogue, succinate) can only bind as part of a ternary complex.     

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.     

Probing the regulatory site of Escherichia coli aspartate transcarbamoylase by site-specific mutagenesis.

The effector binding site of Escherichia coli aspartate transcarbamoylase, composed of the triphosphate and ribose-base subsites, is located on the regulatory (r) chains of the enzyme. In order to probe the function of amino acid side chains at this nucleotide triphosphate site, site-specific mutagenesis was used to create three mutant versions of the enzyme. On the basis of the three-dimensional structure of the enzyme with CTP bound, three residues were selected. Specifically, Arg-96r was replaced with Gln, and His-20r and Tyr-89r were both replaced with Ala. Analyses of these mutant enzymes indicate that none of these substitutions significantly alter the catalytic properties of the enzyme. However, the mutations at His-20r and Tyr-89r produced altered response to the regulatory nucleotides. For the His-20r----Ala enzyme, the affinities of the enzyme for ATP and CTP are reduced 40-fold and 10-fold, respectively, when compared with the wild-type enzyme. Furthermore, CTP is able to inhibit the His-20r----Ala enzyme 40% more than the wild-type enzyme. In the case of the Tyr-89r----Ala enzyme. ATP can increase the mutant enzyme's activity 181% compared to 157% for the wild-type enzyme, while simultaneously the affinity of this enzyme for ATP decreases about 70%. These results suggest that Tyr-89r does have an indirect role in the discrimination between ATP and CTP. The His-20r----Ala enzyme shows no UTP synergistic inhibition in the presence of CTP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coupling of homotropic and heterotropic interactions in Escherichia coli aspartate transcarbamylase

Several types of conditions allow the disconnection of homotropic and heterotropic interactions in Escherichia coli aspartate transcarbamylase. A model that includes a concerted gross conformational change corresponding to the homotropic cooperative interactions between the catalytic sites and local “site by site” effects promoted by the effectors accounts for this disconnection as well as for the other known properties of the enzyme. However, the substrate concentration influences the extent of stimulation and feedback inhibition of the catalytic activity by the effectors. This result is explained by assuming that these effectors promote a “primary effect”, which is exerted locally “site by site”, and a “secondary effect”, which is mediated by the substrate. As predicted by the model, relaxed (R) forms of the enzyme show only the primary effect. In addition 2-ThioU-aspartate transcarbamylase, a modified form of the enzyme in which the homotropic cooperative interactions between the catalytic sites are selectively abolished, shows the same heterogeneity in CTP binding sites as normal aspartate transcarbamylase.