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.     

Effectors of Escherichia coli aspartate transcarbamoylase differentially perturb aspartate binding rather than the T-R transition

New systematic methods developed for equilibrium isotope exchange kinetics have been used to analyze the effects of activator ATP and inhibitor CTP with Escherichia coli aspartate transcarbamoylase. This indepth approach requires (a) variation of [modifier] with fixed subsaturating levels of substrates, and (b) variation of at least three combinations of reactant-product pairs in constant ratio at equilibrium: [A,B,P,Q], [A,P], and [B,Q] with the co-substrates held constant, in the presence and absence of added modifier. Both ATP and CTP had much stronger effects on the [14C]Asp in equilibrium C-Asp exchange rate than on [32P]C-P in equilibrium Pi. The bisubstrate analog N-phosphonacetyl-L-aspartate activated, then inhibited, Asp in equilibrium C-Asp more strongly than C-P in equilibrium Pi. N-Phosphonacetyl-L-aspartate gave complete (100%) inhibition, whereas CTP inhibition of either exchange was only partial. Substrate saturation curves in the presence and absence of effectors indicate that ATP and CTP perturb the observed values of Rmax and S0.5 in different fashions without appreciably changing the observed Hill number. Computer simulations indicate that the primary site of ATP and CTP action is the association rate for Asp, not the allosteric T-R transition. This finding is substantiated by previous studies in which modified aspartate transcarbamoylase had lost cooperative Asp binding without loss of sensitivity to effectors, or in which sensitivity to one effector could be deleted selectively. The present results, with newly devised computer simulation and analysis methods, illustrate the usefulness of equilibrium isotope exchange kinetic probes for providing unique insights to enzyme regulatory mechanisms, to define exactly which steps are altered in a given kinetic mechanism.     

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.     

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

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

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.     

Effects of replacement of active site residue glutamine 231 on activity and allosteric properties of aspartate transcarbamoylase.

Since crystallographic studies on Escherichia coli aspartate transcarbamoylase (ATCase) indicate that Gln 231 is in the active site of the enzyme and participates in the binding of the substrate, aspartate, it seemed of interest to examine mutant enzymes in which Gln 231 was replaced by Asn or Ile. The two mutant forms containing amino acid substitutions were characterized by a combination of steady-state kinetics, hydrodynamic measurements, and equilibrium ligand binding techniques. Both mutant forms exhibited a dramatic reduction in the affinity of the protein for substrates and substrate analogues as well as a very large decrease in catalytic activity. Moreover, the amino acid substitutions introduced within the active site of the enzyme led to unusual allosteric properties in the mutant enzymes. Although the bisubstrate analogue N-(phosphonoacetyl)-L-aspartate promotes the characteristic global conformational change in the mutant forms that is observed with the wild-type enzyme, the combination of substrate and substrate analogue does not. Cooperativity with respect to substrate binding is largely reduced compared to wild-type ATCase. Also, the effector molecules ATP and CTP which bind to the regulatory chains have dramatic effects on the activity of the mutant enzymes containing replacements for Gln 231 in the catalytic chains. In stark contrast to the wild-type enzyme, in which effects of nucleotides are manifested primarily by changes in the K0.5 of the enzyme, ATP and CTP have large effects on the Vmax of the mutant enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lysine-60 in the regulatory chain of Escherichia coli aspartate transcarbamoylase is important for the discrimination between CTP and ATP

Lysine-60 in the regulatory chain of aspartate transcarbamoylase has been changed to an alanine by site-specific mutagenesis. The resulting enzyme exhibits activity and homotropic cooperativity identical with those of the wild-type enzyme. The substrate concentration at half the maximal observed specific activity decreases from 13.3 mM for the wild-type enzyme to 9.6 mM for the mutant enzyme. ATP activates the mutant enzyme to the same extent that it does the wild-type enzyme, but the concentration of ATP required to reach half of the maximal activation is reduced approximately 5-fold for the mutant enzyme. CTP at a concentration of 10 mM does not inhibit the mutant enzyme, while under the same conditions CTP at concentrations less than 1 mM will inhibit the wild-type enzyme to the maximal extent. Higher concentrations of CTP result in some inhibition of the mutant enzyme that may be due either to hetertropic effects at the regulatory site or to competitive binding at the active site. UTP alone or in the presence of CTP has no effect on the mutant enzyme. Kinetic competition experiments indicate that CTP is still able to displace ATP from the regulatory sites of the mutant enzyme. Binding measurements by equilibrium dialysis were used to estimate a lower limit on the dissociation constant for CTP binding to the mutant enzyme (greater than 1 x 10(-3) M). Equilibrium competition binding experiments between ATP and CTP verified that CTP still can bind to the regulatory site of the enzyme. For the mutant enzyme, CTP affinity is reduced approximately 100-fold, while ATP affinity is increased by 5-fold.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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

Replacement of Asp-162 by Ala prevents the cooperative transition by the substrates while enhancing the effect of the allosteric activator ATP on E. coli aspartate transcarbamoylase

The available crystal structures of Escherichia coli aspartate transcarbamoylase (ATCase) show that the conserved residue Asp-162 from the catalytic chain interacts with essentially the same residues in both the T- and R-states. To study the role of Asp-162 in the regulatory properties of the enzyme, this residue has been replaced by alanine. The mutant D162A shows a 7700-fold reduction in the maximal observed specific activity, a twofold decrease in the affinity for aspartate, a loss of homotropic cooperativity, and decreased activation by the nucleotide effector adenosine triphosphate (ATP) compared with the wild-type enzyme. Small-angle X-ray scattering (SAXS) measurements reveal that the unliganded mutant enzyme adopts the T-quaternary structure of the wild-type enzyme. Most strikingly, the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA) is unable to induce the T to R quaternary structural transition, causing only a small alteration of the scattering pattern. In contrast, addition of the activator ATP in the presence of PALA causes a significant increase in the scattering amplitude, indicating a large quaternary structural change, although the mutant does not entirely convert to the wild-type R structure. Attempts at modeling this new conformation using rigid body movements of the catalytic trimers and regulatory dimers did not yield a satisfactory solution. This indicates that intra- and/or interchain rearrangements resulting from the mutation bring about domain movements not accounted for in the simple model. Therefore, Asp-162 appears to play a crucial role in the cooperative structural transition and the heterotropic regulatory properties of ATCase.     

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.     

Different amino acid substitutions at the same position in the nucleotide-binding site of aspartate transcarbamoylase have diverse effects on the allosteric properties of the enzyme

Site-directed mutagenesis was used to determine how the allosteric properties of aspartate transcarbamoylase (ATCase) are affected by amino acid replacements in the nucleotide binding region of the regulatory polypeptide chains. Amino acid substitutions were made for both Lys-60 and Lys-94 in the regulatory chain since those residues have been implicated by x-ray diffraction studies, chemical modification experiments, and site-directed mutagenesis as playing a role in binding CTP and ATP. Lys-60 was replaced by His, Arg, Gln, and Ala, and Lys-94 was changed to His. These mutant forms of ATCase exhibit bewildering changes in the allosteric properties compared to the wild-type enzyme as well as altered affinities for the nucleotide effectors. The enzyme containing His-60 lacks both homotropic and heterotropic effects and exhibits no detectable binding of nucleotides. In contrast, the holoenzymes containing either Gln-60 or Arg-60 retain both homotropic and heterotropic effects. Replacement of Lys-60 by Ala yields a derivative exhibiting altered heterotropic effects involving insensitivity to CTP and activation by ATP. The mutant enzyme containing His-94 in place of Lys exhibits cooperativity with reduced affinity for nucleotides. The multiple substitutions at Lys-60 in the nucleotide binding region of the regulatory chains of ATCase demonstrate that different amino acids in the same location can alter indirectly the delicate balance of interactions responsible for the allosteric properties of ATCase. The studies show that it is hazardous and frequently unwarranted from single amino acid replacements of a specific residue to attribute to that residue the properties observed for the wild-type enzyme.     

A fluorescent probe-labeled Escherichia coli aspartate transcarbamoylase that monitors the allosteric conformational state

A new system has been developed capable of monitoring conformational changes of the 240s loop of aspartate transcarbamoylase, which are tightly correlated with the quaternary structural transition, with high sensitivity in solution. Pyrene, a fluorescent probe, was conjugated to residue 241 in the 240s loop of aspartate transcarbamoylase to monitor changes in conformation by fluorescence spectroscopy. Pyrene maleimide was conjugated to a cysteine residue on the 240s loop of a previously constructed double catalytic chain mutant version of the enzyme, C47A/A241C. The pyrene-labeled enzyme undergoes the normal T to R structural transition, as demonstrated by small-angle x-ray scattering. Like the wild-type enzyme, the pyrene-labeled enzyme exhibits cooperativity toward aspartate, and is activated by ATP and inhibited by CTP at subsaturating concentrations of aspartate. The binding of the bisubstrate analogue N-(phosphonoacetyl)-l-aspartate (PALA), or the aspartate analogue succinate, in the presence of saturating carbamoyl phosphate, to the pyrenelabeled enzyme caused a sigmoidal change in the fluorescence emission. Saturation with ATP and CTP (in the presence of either subsaturating amounts of PALA or succinate and carbamoyl phosphate) caused a hyperbolic increase and decrease, respectively, in the fluorescence emission. The half-saturation values from the fluorescence saturation curves and kinetic saturation curves were, within error, identical. Fluorescence and small-angle x-ray scattering stopped-flow experiments, using aspartate and carbamoyl phosphate, confirm that the change in excimer fluorescence and the quaternary structure change correlate. These results in conjunction with previous studies suggest that the allosteric transition involves both global and local conformational changes and that the heterotropic effect of the nucleotides may be exerted through local conformational changes in the active site by directly influencing the conformation of the 240s loop.     

Heterotropic effectors promote a global conformational change in aspartate transcarbamoylase

The sigmoidal dependence of activity on substrate concentration exhibited by the regulatory enzyme aspartate transcarbamoylase (ATCase) of Escherichia coli is generally attributed to a ligand-promoted change in the quaternary structure of the enzyme. Although a global conformational change in ATCase upon the binding of ligands to some of the six active sites is well documented, a corresponding alteration in the structure of the wild-type enzyme upon the addition of the inhibitor, CTP, or the activator, ATP, has not been detected. Such evidence is essential for testing whether heterotropic, as well as homotropic, effects can be accounted for quantitatively in terms of coupled equilibria involving a conformational change in the enzyme and preferential binding of ligands to one conformation or the other. This evidence has now been obtained with a mutant form of ATCase in which Lys 143 in the regulatory chain was replaced by Ala, thereby perturbing interactions at the interface between the regulatory and catalytic chains in the enzyme and destabilizing the low-activity, compact (T) conformation relative to the high-activity, swollen (R) state. Difference sedimentation velocity experiments involving measurements of the changes caused by the binding of the bisubstrate analogue N-(phosphonacetyl)-L-aspartate demonstrated that the sedimentation coefficient of the mutant enzyme was intermediate between that observed for the T and R states of wild-type ATCase. We interpret the results as indicating that the [T]/[R] ratio in phosphate buffer at pH 7.0 is reduced from about 2 X 10(2) for the wild-type enzyme to 2.7 for r143Ala ATCase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Catalysis of partial reactions of ATP synthesis by beef heart mitochondrial adenosine triphosphatase

We have found that when the ATP hydrolysis activity of beef heart mitochondrial adenosine triphosphatase (F1) is eliminated by either cold treatment or chemical modification, the enzyme attains the ability to catalyze the Pi in equilibrium ATP exchange reaction. The ATP hydrolysis activity of isolated F1 was lost upon chemical modification by phenyglyoxal, butanedione, or 7-chloro-4-nitrobenzene-2-oxa-1,3-diazole. The F1 thus chemically modified was able to catalyze an ADP-dependent Pi in equilibrium ATP exchange reaction. In addition F1 that had been cold-treated to eliminate ATP hydrolysis activity, also catalyzed the Pi in equilibrium ATP exchange reaction. The Pi in equilibrium ATP exchange catalyzed by modified F1 was shown to be totally inhibited by the F1-specific antibiotic efrapeptin. We have previously shown that isolated beef heart mitochondrial ATPase will catalyze the formation of a transition state analog of the ATP synthesis reaction (Bossard, M. J., Vik, T. A., and Schuster, S. M. (1980) J. Biol. Chem. 255, 5342-5346). While the F1-catalyzed ATP hydrolysis activity was lost rapidly upon chemical modification or cold treatment, the ability of the enzyme to produce Pi . adenosine 5'-diphosphate (chromium(III) salt) from phosphate and monodentate adenosine 5'-diphosphate (chromium(III) salt) was unimpaired. The implications of these data with regard to the mechanism of ATP synthesis are discussed.     

Identification of Ser424 as the protein kinase A phosphorylation site in CTP synthetase from Saccharomyces cerevisiae.

The URA7-encoded CTP synthetase [EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)] in the yeast Saccharomyces cerevisiae is phosphorylated on a serine residue and stimulated by cAMP-dependent protein kinase (protein kinase A) in vitro. In vivo, the phosphorylation of CTP synthetase is mediated by the RAS/cAMP pathway. In this work, we examined the hypothesis that amino acid residue Ser424 contained in a protein kinase A sequence motif in the URA7-encoded CTP synthetase is the target site for protein kinase A. A CTP synthetase synthetic peptide (SLGRKDSHSA) containing the protein kinase A motif was a substrate (Km = 30 microM) for protein kinase A. This peptide also inhibited (IC50 = 45 microM) the phosphorylation of purified wild-type CTP synthetase by protein kinase A. CTP synthetase with a Ser424 --> Ala (S424A) mutation was constructed by site-directed mutagenesis. The mutated enzyme was not phosphorylated in response to the activation of protein kinase A activity in vivo. Purified S424A mutant CTP synthetase was not phosphorylated and stimulated by protein kinase A. The S424A mutant CTP synthetase had reduced Vmax and elevated Km values for ATP and UTP when compared with the protein kinase A-phosphorylated wild-type enzyme. The specificity constants for ATP and UTP for the S424A mutant CTP synthetase were 4.2- and 2.9-fold lower, respectively, when compared with that of the phosphorylated enzyme. In addition, the S424A mutant enzyme was 2.7-fold more sensitive to CTP product inhibition when compared with the phosphorylated wild-type enzyme. These data indicated that the protein kinase A target site in CTP synthetase was Ser424 and that the phosphorylation of this site played a role in the regulation of CTP synthetase activity.     

Tyrosine-7 in human class Pi glutathione S-transferase is important for lowering the pKa of the thiol group of glutathione in the enzyme-glutathione complex.

Previously, we reported the importance of Tyr7 for the catalytic activity of human class Pi glutathione S-transferase [Kong et al. (1992) Biochem. Biophys. Res. Comm., 182, 1122]. As an extension of this study, we investigated the pH dependence of kinetic parameters of the wild-type enzyme and the Y7F mutant. The replacement of Tyr7 with phenylalanine was found to alter the pH dependence of Vmax and Vmax/KmCDNB of the enzyme for conjugation of GSH with 1-chloro-2,4-dinitrobenzene (CDNB). The pKa of the thiol of GSH in the wild-type enzyme-GSH complex was estimated to be about 2.4 pK units lower than that in the Y7F-GSH complex. Tyr7 is thus considered to be important for catalytic activity in lowering the pKa of the thiol of GSH in the enzyme-GSH complex.     

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)     

Escherichia coli aspartate transcarbamylase: a novel marker for studies of gene amplification and expression in mammalian cells.

Eucaryotic expression vectors containing the Escherichia coli pyrB gene (pyrB encodes the catalytic subunit of aspartate transcarbamylase [ATCase]) and the Tn5 phosphotransferase gene (G418 resistance module) were transfected into a mutant Chinese hamster ovary cell line possessing a CAD multifunctional protein lacking ATCase activity. G418-resistant transformants were isolated and analyzed for ATCase activity, the ability to complement the CAD ATCase defect, and the ability to resist high concentrations of the ATCase inhibitor N-(phosphonacetyl)-L-aspartate (PALA) by amplifying the donated pyrB gene sequences. We report that bacterial ATCase is expressed in these lines, that it complements the CAD ATCase defect in trans, and that its amplification engenders PALA resistance. In addition, we derived rapid and sensitive assay conditions which enable the determination of bacterial ATCase enzyme activity in the presence of mammalian ATCase.     

Crystal structure of Sulfolobus acidocaldarius aspartate carbamoyltransferase in complex with its allosteric activator CTP

Aspartate carbamoyltransferase (ATCase) is a paradigm for allosteric regulation of enzyme activity. B-class ATCases display very similar homotropic allosteric behaviour, but differ extensively in their heterotropic patterns. The ATCase from the thermoacidophilic archaeon Sulfolobus acidocaldarius, for example, is strongly activated by its metabolic pathway′s end product CTP, in contrast with Escherichia coli ATCase which is inhibited by CTP. To investigate the structural basis of this property, we have solved the crystal structure of the S. acidocaldarius enzyme in complex with CTP. Structure comparison reveals that effector binding does not induce similar large-scale conformational changes as observed for the E. coli ATCase. However, shifts in sedimentation coefficients upon binding of the bi-substrate analogue PALA show the existence of structurally distinct allosteric states. This suggests that the so-called “Nucleotide-Perturbation model” for explaining heterotropic allosteric behaviour, which is based on global conformational strain, is not a general mechanism of B-class ATCases.     

Dissecting enzyme regulation by multiple allosteric effectors: nucleotide regulation of aspartate transcarbamoylase

The enzyme aspartate transcarbamoylase (ATCase, EC 2.1.3.2 of Escherichia coli), which catalyzes the committed step of pyrimidine biosynthesis, is allosterically regulated by all four ribonucleoside triphosphates (NTPs) in a nonlinear manner. Here, we dissect this regulation using the recently developed approach of random sampling-high-dimensional model representation (RS-HDMR). ATCase activity was measured in vitro at 300 random NTP concentration combinations, each involving (consistent with in vivo conditions) all four NTPs being present. These data were then used to derive a RS-HDMR model of ATCase activity over the full four-dimensional NTP space. The model accounted for 90% of the variance in the experimental data. Its main elements were positive ATCase regulation by ATP and negative by CTP, with the negative effects of CTP dominating the positive ones of ATP when both regulators were abundant (i.e., a negative cooperative effect of ATP x CTP). Strong sensitivity to both ATP and CTP concentrations occurred in their physiological concentration ranges. UTP had only a slight effect, and GTP had almost none. These findings support a predominant role of CTP and ATP in ATCase regulation. The general approach provides a new paradigm for dissecting multifactorial regulation of biological molecules and processes.