Modes of modifier action in E. coli aspartate transcarbamylase

The observed patterns for inhibition by CTP and succinate of equilibrium exchange kinetics with native aspartate transcarbamylase (E. coli) are consistent with an ordered substrate-binding system in which aspartate binds after carbamyl phosphate, and phosphate is released after carbamyl aspartate. ATP selectively stimulates Asp carbamyl-Asp exchange, but not carbamyl phosphate P_i. Initial velocity studies at 5 °, 15 °, and 35 °C were carried out, using modifiers as perturbants of the system. Modifiers alter the Hill n and S_0.5 for aspartate, most markedly at 15 °C but less so at the other temperatures. ATP does increase V under saturating substrate conditions, and substrate inhibition is observed for aspartate. ATP does not make the Hill n = 1 at any temperature. It is proposed that CTP and ATP act by separate mechanisms, not by simply perturbing in opposite directions the equilibrium for aspartate binding. ATP appears to act to increase the rate of aspartate association and dissociation, whereas CTP induces an intramolecular competitive effect in the protein.     

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.     

Use of analytical gel chromatography to analyze tertiary and quaternary structural changes in E. coli aspartate transcarbamylase

E. coli aspartate transcarbamylase (ATCase) is a large (310 kDa) protein that undergoes major changes in quaternary structure when substrates and regulatory nucleotides bind. We have used analytical gel chromatography to detect quaternary structure changes in both the holoenzyme and its catalytic subunit (c3), to characterize the quaternary structure of single site mutant proteins and to monitor urea-induced dissociation and unfolding of c3. Binding of the bisubstrate analog PALA (N-(phosphonacetyl)-L-aspartate) to ATCase and c3 has been shown to alter s20.w by -3.3% and + 1.4%, respectively [Howlett, G.J. and Schachman, H.K. (1977), Biochemistry 23, 5077-5083]. The corresponding changes in the chromatographic partition coefficient (sigma) are -2.6 +/- 0.3% and 5.5 +/- 1.9% on Sephacryl S400HR and S200, respectively. Partition coefficients of mutant ATCases with single site mutations in the c chain differ from those of the wild-type protein by +/- 0.5% in small zone experiments; for example, mutations Arg 269----Gly and Glu 239----Gln alter the partition coefficient by 0.4% and -0.5%, respectively. The partition coefficient of mutant Glu 50----Gln is identical to the wild type enzyme. In the presence of saturating PALA, partition coefficients of Glu 50----Gln and Arg 269----Gly, but not Glu 239----Gln are identical to those of the wild type. Results for Glu 239----Gln are consistent with measurements of activity, small angle X-ray scattering and sedimentation coefficient that indicate that mutations at this site shift the quaternary structure towards the R state [Ladjimi and Kantrowitz (1988), Biochemistry 27, 276-83; Vachette and Hervé, cited by Kantrowitz and Lipscomb (1988), Science 241, 669-674; Newell and Schachman (1988), FASEB J. 2, A551]. Results for Glu 50----Gln are also consistent with measurements of activity (Ladjimi et al. (1988), Biochemistry 27, 268-276). The changes in tertiary and quaternary structure that result from urea-induced denaturation of c3 result in larger changes in the partition coefficient. Dissociation into folded monomers in 1-1.75 M urea is accompanied by a 4.6% increase in partition coefficient, while denaturation at greater than 5 M urea gives rise to a 43% decrease on S-300 Sephacryl. The bisubstrate analog PALA suppresses dissociation and increases the cooperativity of the unfolding reaction.     

Allosteric activation of aspartate transcarbamylase with a fluorescent nucleotide analogue: linear-benzo-ATP.

The interaction of Escherichia coli aspartate transcarbamylase with linear-benzo-ATP has been investigated by means of fluorescence spectroscopy. The fluorescent nucleotide analogue activates the enzyme to the same extent as ATP. Fluorescence polarization has been used to determine the association constant of lin-benzo-ATP with aspartate transcarbamylase (ATCase) which is 5 X 10(-3) M-1 at pH 8.7, at 4 degrees C, assuming six binding sites. This association constant is similar to those previously obtained for ATP at a variety of temperatures, buffers, and pH. The fluorescence emission of lin-benzo-ATP is not quenched when bound to ATCase, which indicates absence of pi interactions between the activator and tyrosyl residues in the protein. These residues have been implicated in the stereochemical mechanism of allosteric interactions in ATCase. Furthermore, this fluorescence behavior implicates hydrogen bond formation between the amino group of lin-benzo-ATP and a nucleophilic center at the enzyme binding site. The fact that lin-benzo-ATP activates ATCase is consistent with a previously published model for nucleotide regulation of the enzyme.     

Kinetic mechanism of native Escherichia coli aspartate transcarbamylase

Equilibrium isotope exchange kinetics have been used to reinvestigate the kinetic mechanism of Escherichia coli aspartate transcarbamylase (aspartate carbamoyl-transferase) at pH 7.0, 30 degrees C. Keq = 5.9 (+/- 0.6) X 10(3), allowing variation of substrate concentrations above and below their Km values in all experiments, a condition not possible at pH 7.8 [F. C. Wedler and F. J. Gasser (1974) Arch. Biochem. Biophys. 163, 57-68]. The rate of the [14C]Asp in equilibrium N-carbamoyl L-aspartate (C-Asp) exchange reaction was five times faster than that of [32P]carbamyl phosphate (C-P) in equilibrium Pi, which argues strongly against the rapid equilibrium random mechanism previously proposed by E. Heyde, A. Nagabhushanam, and J. F. Morrison [Biochemistry 12, 4718-4726 (1973]. Substrate concentrations were varied either as reactant-product pairs (holding the other pair constant) or together simultaneously in constant ratio at equilibrium. The resulting kinetic saturation patterns were most consistent with a preferred order random kinetic mechanism, with C-P binding prior to Asp and with C-Asp being released before Pi. Weak inhibition effects at high substrate levels could be accounted for by multiple weak dead-end complexes or ionic strength effects. Computer-based simulations have led to a set of rate constants that fit the experimental data, are in agreement with rate constants measured previously by pre-steady-state methods, and predict the correct initial velocities in the forward and reverse directions. Simulations also show that rate constants consistent with any of the various alternative mechanisms do not provide good fit to the experimental data. A model for the kinetic mechanism is considered, in which the binding of Asp prior to C-P may restrict access of C-P to the active site, but C-P binding prior to Asp potentiates the enzyme for the allosteric (T-R) transition, centered entirely upon the Asp binding process.     

Ordered substrate binding and evidence for a thermally induced change in mechanism for E. coli aspartate transcarbamylase

Isotopic exchange kinetics at equilibrium for E. coli native aspartate transcarbamylase at pH 7.8, 30 °C, are consistent with an ordered BiBi substrate binding mechanism. Carbamyl phosphate binds before l-Asp, and carbamyl-aspartate is released before inorganic phosphate. The rate of [¹⁴C]Asp C-Asp exchange is much faster than [³²P]carbamyl phosphate P_i exchange. Phosphate, and perhaps carbamyl phosphate, appears to bind at a separate modifier site and prevent dissociation of active-site bound P_i or carbamyl phosphate. Initial velocity studies in the range of 0–40 °C reveal a biphasic Arrhenius plot for native enzyme: E_a (>15 °C) = 6.3 kcal/ mole and E_a (<15 °C) = 22.1 kcal/mole. Catalytic subunits show a monophasic plot with E_a ? 20.2 kcal/mole. This, with other data, suggests that with native enzyme a conformational change accompanying aspartate association contributes significantly to rate limitation at t > 15 °C, but that catalytic steps become definitively slower below 15 °C. Model kinetics are derived to show that this change in mechanism at low temperature can force an ordered substrate binding system to produce exchange-rate patterns consistent with a random binding system with all exchange rates equal. The nonlinear Arrhenius plot also has important consequences for current theories of catalytic and regulatory mechanisms for this enzyme.     

Electrostatic interactions in the assembly of Escherichia coli aspartate transcarbamylase

Although ionizable groups are known to play important roles in the assembly, catalytic, and regulatory mechanisms of Escherichia coli aspartate transcarbamylase, these groups have not been characterized in detail. We report the application of static accessibility modified Tanford-Kirkwood theory to model electrostatic effects associated with the assembly of pairs of chains, subunits, and the holoenzyme. All of the interchain interfaces except R1-R6 are stabilized by electrostatic interactions by -2 to -4 kcal-m-1 at pH 8. The pH dependence of the electrostatic component of the free energy of stabilization of intrasubunit contacts (C1-C2 and R1-R6) is qualitatively different from that of intersubunit contacts (C1-C4, C1-R1, and C1-R4). This difference may allow the transmission of information across subunit interfaces to be selectively regulated. Groups whose calculated pK or charge changes as a result of protein-protein interactions have been identified and the results correlated with available information about their function. Both the 240s loop of the c chain and the region near the Zn(II) ion of the r chain contain clusters of ionizable groups whose calculated pK values change by relatively large amounts upon assembly. These pK changes in turn extend to regions of the protein remote from the interface. The possibility that networks of ionizable groups are involved in transmitting information between binding sites is suggested.     

Effects of the T→R transition on the electrostatic properties of E. coli aspartate transcarbamylase

Aspartate transcarbamylase is a large (310 kD), multisubunit protein that binds substrates cooperatively and undergoes a large change in quaternary structure when substrates bind. The forces that drive this transition are poorly understood. We evaluated the electrostatic component of these forces by using finite difference and multigrid methods to solve the nonlinear Poisson-Boltzmann equation for complexes of the enzyme with several substrates and substrate analogs. The results have been compared with calculations for the unliganded protein. While pK_½ values of most ionizable residues fall within 3 pH units of values for model compounds, 31 have pK_½ values that fall outside the range 0–17. Many of these residues are at the active site, where they interact with the highly charged substrate, in the 80s loop or 240s loop or interact with these loops. The pK_½ values of eight ionizable residues related by the twofold molecular axes differ by more than 3 pH units, providing additional evidence for asymmetry within the crystal. As in the unliganded structure, a set of residues forms a network in which ionizable groups with W_ij values greater than 2 kcal-m^-1 are separated by distances greater than 5 Å. Some residues participate in this network in both the unliganded and N-phosphonacetyl-L-aspartate (PALA)-liganded structure, while others are found in only one structure. The network is more extensive in the PALA-liganded structure than in the unliganded structure, but consists of two separate networks in the two halves of the molecule. Proteins 32:200–210, 1998. © 1998 Wiley-Liss, Inc.     

Kinetic mechanism of catalytic subunits (c3) of E. coli aspartate transcarbamylase at pH 7.0

In contrast to holo-enzyme (c6r6), catalytic subunits (c3) of Escherichia coli aspartate transcarbamylase (carbamoyl-phosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) do not exhibit allosteric interactions or inhibition effects that complicate kinetic investigations of substrate binding order. Equilibrium isotope-exchange kinetic probes of c3 at pH 7.0 and 30 degrees C produced kinetic saturation patterns consistent with a strongly preferred order random kinetic mechanism, in which carbamoyl phosphate binds prior to aspartate and carbamoyl aspartate is released before Pi. Weak substrate inhibition effects observed with c6r6 did not occur with c3, possibly due to decreased affinity for ligands at the dianion inhibition site.     

Comparison of van 't Hoff and calorimetrically determined enthalpies of binding of N-phosphonacetyl-L-aspartate to E. coli aspartate transcarbamylase.

A comparison has been made of the values obtained by direct calorimetric measurements and van 't Hoff analysis, under similar conditions, for the enthalpy of binding of the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA) to E. coli aspartate transcarbamylase and its catalytic subunit. In the case of the catalytic subunit, data were obtained at both saturating and non-saturating concentrations of L-Asp, and at two ionic strengths. Despite a 1000-fold difference in protein concentrations, and the obligatory omission of carbamyl phosphate in the calorimetric experiments, the values obtained by the two methods are shown to agree to within 15% when appropriate corrections are made. These results suggest that subunit dissociation is not a significant factor at the low protein concentrations used in the van 't Hoff analysis, and, conversely, that aggregation of the protein is negligible at the high protein concentrations used in the calorimetric experiments. They also imply that, at pH 8.3, the enthalpic difference between the two conformational states of the enzyme which exist in the presence and absence of substrates is less than 2.5 kcal/mol. In addition, the trends in the three sets of data for the catalytic subunit indicate that ionic bonds are involved in binding PALA to the active site, and that non-productive binding by L-Asp is negligible under these experimental conditions.     

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.     

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.     

Importance of domain closure for homotropic cooperativity in Escherichia coli aspartate transcarbamylase

The importance of the interdomain bridging interactions observed only in the R-state structure of Escherichia coli aspartate transcarbamylase between Glu-50 of the carbamoyl phosphate domain with both Arg-167 and Arg-234 of the aspartate domain has been investigated by using site-specific mutagenesis. Two mutant versions of aspartate transcarbamylase were constructed, one with alanine at position 50 (Glu-50----Ala) and the other with aspartic acid at position 50 (Glu-50----Asp). The alanine substitution totally prevents the interdomain bridging interactions, while the aspartic acid substitution was expected to weaken these interactions. The Glu-50----Ala holoenzyme exhibits a 15-fold loss of activity, no substrate cooperativity, and a more than 6-fold increase in the aspartate concentration at half the maximal observed specific activity. The Glu-50----Asp holoenzyme exhibits a less than 3-fold loss of activity, reduced cooperativity for substrates, and a 2-fold increase in the aspartate concentration at half the maximal observed specific activity. Although the Glu-50----Ala enzyme exhibits no homotropic cooperativity, it is activated by N-(phosphonoacetyl)-L-aspartate (PALA). As opposed to the wild-type enzyme, the Glu-50----Ala enzyme is activated by PALA at saturating concentrations of aspartate. At subsaturating concentrations of aspartate, both mutant enzymes are activated by ATP, but are inhibited less by CTP than is the wild-type enzyme. At saturating concentrations of aspartate, the Glu-50----Ala enzyme is activated by ATP and inhibited by CTP to an even greater extent than at subsaturating concentrations of aspartate.(ABSTRACT TRUNCATED AT 250 WORDS)     

A kinetic model of cooperativity in aspartate transcarbamylase.

A relatively simple kinetic model is proposed to account simultaneously for data on the binding of carbamyl phosphate and succinate to aspartate trans carbamylase (ATCase), and for the relaxation spectrum associated with this binding. The model also accounts for measurements of the initial velocity of the reaction of ATCase with respect to aspartate and carbamyl phosphate. The principal assumption made is that ATCase consists of three identical noninteracting cooperative dimers. Ordered binding and both sequential and concerted conformational changes in the dimers are needed to account for the properties of ATCase. The values of the parameters of this model can be determined by fitting to existing experimental evidence. Various new quantitative predictions are made that can serve as additional tests of the proposed theory.     

The effect of pH on the cooperative behavior of aspartate transcarbamylase from Escherichia coli.

Saturation curves of activity versus concentration were determined for aspartate transcarbamylase from Escherichia coli (EC 2.1.3.2) for the substrate L-aspartate at saturating carbamyl phosphate (4.8 mM) in buffered solution at pH values from 6.0 to 12.0. Hill coefficients were obtained from the sigmoidal curves. At pH values from 7.8 to 9.1, where substrate inhibition causes difficulties in the Hill approximation, our kinetic scheme includes substrate inhibition and residual activity in the abortive enzyme-substrate complex. The plot of Hill coefficient versus pH has pKalpha values of 7.4 and 9.8 at the half-maximum positions of the curve which has a plateau from pH 8.1 to 9.1. These pKalpha values may be associated with functional groups involved in the allosteric transition which activates the enzyme. A plot of [S]0.5 versus pH shows a pKalpha of 8.5, which may belong to a residue either at or near the aspartate binding site. At 50 mM aspartate concentration the pH-rate profile shows maxima at pH values of 8.8 and 10.0 (cf. Weitzman, P.D.J., and Wilson, I.B.(1966)J. Biol. Chem. 2418 5481-5488, who used 100 mM aspartate). However, when the pH-dependent substrate inhibition is included, the calculated Vmax--H curve is bell-shaped like that of the isolated catalytic subunit.