Differential scanning calorimetric study of the thermal denaturation of aspartate transcarbamoylase of Escherichia coli

The thermal denaturation of Escherichia coli aspartate transcarbamoylase (c6r6) in the absence and presence of various ligands has been studied by means of high-sensitivity differential scanning calorimetry (DSC). As previously reported [Vickers, K.P., Donovan, J.W., & Schachman, H.K. (1978) J. Biol. Chem. 253, 8493-8498], the denaturational endotherm consists of two peaks, the lower of which is due to denaturation of the three regulatory, r2, subunits while the upper involves the two catalytic, c3, subunits. The temperature of maximal excess apparent specific heat, tm, of the lower peak is raised from the value of 51.4 degrees C for the isolated subunit to 66.8 degrees C as a result of subunit interactions, whereas tm for the c3 peak is essentially the same in the isolated subunit and in the holoenzyme, indicating that the denatured r2 subunits do not interact with the c3 subunits. The total specific denaturational enthalpy for c6r6, 4.83 +/- 0.16 cal g-1, is significantly larger than the weighted mean, 4.08 cal g-1, of the enthalpies for c3 and r2. The fact that no endotherm is observed when previously scanned protein is rescanned indicates that the denaturation is irreversible, as is also the case with the r2 and c3 subunits. Empirical justification for analyzing the data in terms of equilibrium thermodynamics is cited. The observed DSC curves can be expressed within experimental uncertainty as the sum of five sequential two-state steps. The value of t 1/2, the temperature of half-completion, for each step increases with increasing protein concentration, indicating that some dissociation of the protein takes place during denaturation.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Site-specific mutation of Tyr240----Phe in the catalytic chain of Escherichia coli aspartate transcarbamylase. Consequences for kinetic mechanism

In the catalytic chain of Escherichia coli aspartate transcarbamylase, Tyr240 helps stabilize the T-state conformation by an intrachain hydrogen bond to Asp271. Changes in kinetic characteristics of ATCase that result from disruption of this bond by site-specific mutation of Tyr240----Phe have been investigated by isotopic exchanges at chemical equilibrium. The Tyr240----Phe (Y240F) mutation caused the rate of the [32P] carbamyl phosphate (C-P) in equilibrium Pi exchange to decrease by 2-8-fold, without altering the [14C]Asp in equilibrium N-carbamyl-L-aspartate (C-Asp) rate. The mutation also caused the S0.5 and Hill nH values to decrease in virtually every substrate saturation experiment. Upon increasing the concentrations of the C-P,Pi or C-P,C-Asp reactant-product pairs, inhibition effects observed with the C-P in equilibrium Pi exchange for wild-type enzyme were not apparent with the Y240F mutant enzyme. In contrast, upon increasing the concentrations of the Asp,C-Asp and Asp,Pi pairs, inhibition effects on C-P in equilibrium Pi observed with wild-type enzyme became stronger with the Y240F mutant enzyme. These data indicate that the Tyr240----Phe mutation alters the kinetic mechanism in two different ways: on the reactant side, C-P binding prior to Asp shifts from preferred to compulsory order, and, on the product side, C-Asp and Pi release changes from preferred to nearly random order. These conclusions were also confirmed on a quantitative basis by computer simulations and fitting of the data, which also produced an optimal set of rate constants for the Y240F enzyme. The Arrhenius plot for wild-type holoenzyme was biphasic, but those for catalytic subunits and Y240F enzyme were linear (monophasic). Taken together, the data indicate that the Tyr240----Phe mutation destabilizes the T-state and shifts the equilibrium for the T-R allosteric transition toward the R-state by increasing the rate of T----R conversion.     

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.     

Regulatory behavior of Escherichia coli aspartate transcarbamylase altered by site-specific mutation of Tyr240----Phe in the catalytic chain

Isotopic exchange kinetics at chemical equilibrium have been used to identify changes in the regulatory properties of aspartate transcarbamylase (ATCase) caused by site-specific mutation of Tyr240----Phe (Y240F) in the catalytic chain. With both wild-type and the mutant enzymes, ATP activates both [14C]Asp in equilibrium N-carbamyl-L-aspartate (C-Asp) and the [32P]carbamyl phosphate (C-P) in equilibrium Pi exchanges. In contrast, with wild-type enzyme, CTP inhibits both exchanges, but with Y240F mutant enzyme CTP inhibits Asp in equilibrium C-Asp exchange and activates C-P in equilibrium Pi exchange. The bisubstrate analog N-(phosphonacetyl-L-aspartate), PALA, activates Asp in equilibrium C-Asp at a lower concentration with the Y240F enzyme, but the extent of activation is decreased, relative to wild-type enzyme. PALA activation of C-P in equilibrium Pi observed with wild-type enzyme disappears completely with the Y240F mutant enzyme. Analysis of perturbations of exchange rates by ATP and CTP were carried out by systematic methods plus computer-based simulations with the ISOBI program. These analyses indicate that (a) ATP increases the rates of association and dissociation for both C-P and Asp, but (b) CTP differentially increases the rate of C-P association to a greater degree than dissociation, but also decreases the rates for Asp association and dissociation in equal proportion. In addition, Arrhenius plots for Y240F ATCase suggest that ATP and CTP act by different mechanisms: ATP increases Vmax (decreases delta G not equal to) uniformly at all temperatures, whereas CTP does not alter either Vmax (delta G not equal to) or the Arrhenius slope (delta H not equal to).     

Differential scanning calorimetric studies on bovine serum albumin: III. Effect of sodium dodecyl sulphate

Measurements of differential scanning calorimetry (d.s.c.) have been made on the complex bovine serum albumin (BSA)--sodium dodecyl sulphate (SDS) under various conditions. There are two peaks P1 and P2 in the d.s.c. curve for BSA at pH 7 and in the absence of NaCl, indicating the presence of the heat-induced transition of BSA. There are three peaks P1, P2 and P3 in the curve for the system with the molar mixing ratio SDS/BSA = 1. With the increase in the amount of SDS, the peak P3 grows at the expense of P1 and P2. There is only a single peak P3 in the curve for the systems SDS/BSA > 7, and no peak at SDS/BSA = 50 and 100. There is a single peak P12 in the curve for BSA at pH 7 and in the presence of 0.05 M NaCl, indicating that the heat-induced transition is suppressed. There are two peaks P12 and P3 for the systems SDS/BSA = 1-5; the area ratio of the peak P3 to P12 increases with the increase in the amount of SDS. There is only a single peak P3 when SDS/BSA > 7, and no peak at SDS/BSA = 50. It is concluded that the peak P3 is a product of SDS regardless of the presence or absence of NaCl. Values of thermal denaturation temperature (Td) and enthalpy (delta H) of thermal denaturation indicate that the complex AD12 (A = BSA, D = SDS) is in the most thermostabilized state.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-resolution differential scanning calorimetric analysis of the subunits of Escherichia coli aspartate transcarbamoylase

The thermal denaturation of the catalytic (c3) and regulatory (r2) subunits of Escherichia coli aspartate transcarbamoylase (c6r6) in the absence and presence of various ligands has been studied by means of highly sensitive differential scanning calorimetry. The denaturation of both types of subunit is irreversible as judged by the facts that the proteins coagulate when heated and that no endotherm is observed when previously scanned protein is rescanned. Despite this apparent irreversibility, there is empirical justification for analyzing the calorimetric data in terms of equilibrium thermodynamics as embodied in the van't Hoff equation. The observed curves of excess apparent specific heat vs. temperature are asymmetric and can be expressed within experimental uncertainty as the sums of sequential two-state steps, a minimum of two steps being required for r2 and three for c3. As previously reported [Vickers, K. P., Donovan, J. W., & Schachman, H. K. (1978) J. Biol. Chem. 253, 8493-8498], the addition of the effectors ATP and CTP raises the denaturation temperature of r2 and lowers that of c3 while the addition of the bisubstrate analogue N-(phosphonoacetyl)-L-aspartate raises the denaturation temperature of c3 and lowers that of r2. These effects vary with ligand concentration in the manner expected from the van't Hoff equation, indicating that they are simply manifestations of Le Chatelier's principle rather than being due to "stabilization" or "destabilization" of the proteins. The denaturational enthalpy is increased in those cases of ligand binding in which the denaturation temperature is increased, because of the contribution from the enthalpy of dissociation of the ligand.     

Ligation alters the pathway of urea-induced denaturation of the catalytic trimer of Escherichia coli aspartate transcarbamylase.

We have examined the pathway and energetics of urea-induced dissociation and unfolding of the catalytic trimer (c3) of aspartate transcarbamylase from Escherichia coli at low temperature in the absence and presence of carbamyl phosphate (CP; a substrate), N-(phosphonacetyl)-L-Asp (PALA; a bisubstrate analog), and 2 anionic inhibitors, Cl- and ATP, by analytical gel chromatography supplemented by activity assays and ultraviolet difference spectroscopy. In the absence of active-site ligands and in the presence of ATP, c3 dissociates below 2 M urea into swollen c chains that then gradually unfold from 2 to 6 M urea with little apparent cooperativity. Linear extrapolation to 0 M urea of free energies determined in 3 independent types of experiments yields estimates for delta Gdissociation at 7.5 degrees C of about 7-10 kcal m-1 per interface. delta Gunfolding of dissociated chains when modeled as a 2-state process is estimated to be very small, on the order of -2 kcal m-1. The data are also consistent with the possibility that the unfolding of the dissociated monomer is a 1-state swelling process. In the presence of the ligands CP and PALA, and in the presence of Cl-, c3 dissociates at much higher urea concentrations, and trimer dissociation and unfolding occur simultaneously and apparently cooperatively, at urea concentrations that increase with the affinity of the ligand.     

Accessory protein function in the DNA polymerase III holoenzyme from E. coli.

DNA polymerases which duplicate cellular chromosomes are multiprotein complexes. The individual functions of the many proteins required to duplicate a chromosome are not fully understood. The multiprotein complex which duplicates the Escherichia coli chromosome, DNA polymerase III holoenzyme (holoenzyme), contains a DNA polymerase subunit and nine accessory proteins. This report summarizes our current understanding of the individual functions of the accessory proteins within the holoenzyme, lending insight into why a chromosomal replicase needs such a complex structure.     

Differential scanning calorimetric studies on bovine serum albumin IV. Effect of anionic surfactants with various lengths of hydrocarbon chain

Using defatted and SH-blocked bovine serum albumin (BSA), measurements of differential scanning calorimetry (DSC) have been made at pH 7 on the complexes of BSA and a series of sodium alkyl sulfates. Alkyl sulfates used were sodium decyl sulfate (SDeS), sodium octyl sulfate (SOS), sodium hexyl sulfate (SHS) and sodium ethyl sulfate (SES). Results obtained were compared with those on the system BSA-sodium dodecyl sulfate (SDS) studied previously. Two peaks P 1 and P2 existed in the DSC curve of BSA. These peaks originate in the heat-induced transition of BSA. The pattern of DSC curve changed with the amount of the ligand added, i.e. with the molar mixing ratio ligand/BSA (1). The change for systems BSA-SDeS, BSA-SOS and BSA-SHS was qualitatively the same as that for the system BSA-SDS (2). Interestingly, SES, which is not a surfactant, interacts with BSA. The change for the system BSA-SES was qualitatively the same as that for the system BSA-Na₂SO₄. All alkyl sulfates suppressed the heat-induced transition at lower concentrations. A linear relationship was obtained for the plots of log(D/A)₁ versus log CMC, where (D/A)₁ is the molar mixing ratio of anionic surfactant (D) to BSA (A) at which the most heat-stable complex is formed. This suggests that the hydrophobic force has a serious effect on the formation of heat-stable complexes.     

Kinetic consequences of site-specific mutation of Glu-239----Gln in E. coli aspartate transcarbamylase: comparison with catalytic subunits and Phe-240 mutant enzyme

The kinetic characteristics of E. coli aspartate transcarbamylase, altered by site-specific mutagenesis of Glu-239----Gln, have been determined by equilibrium isotope-exchange kinetics and compared to the wild-type system. In wild-type enzyme, residue Glu-239 helps to stabilize the T-state structure by multiple bonding interactions with Tyr-165 and Lys-164 across the c1-c4 subunit interface; upon conversion to the R-state, these bonds are re-formed within c-chains. Catalysis of both the [14C]Asp in equilibrium C-Asp and [32P]ATP in equilibrium Pi exchanges by mutant enzyme occurs at rates comparable to those for wild-type enzyme. Saturation with different reactant/product pairs produced kinetic patterns consistent with strongly preferred order binding of carbamyl-P prior to Asp and carbamyl-Asp release before Pi. The kinetics for the Gln-239 mutant enzyme resemble those observed for catalytic subunits (c3), namely a R-state enzyme (Hill coefficient nH = 1.0) and Km (Asp) approximately equal to 6 mM. The Glu-239----Gln mutation appears to destablize both the T- and R-states, whereas the Tyr-240----Phe mutation destablizes only the T-state.     

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.     

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.     

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'.