Catalytic Subunit Of Aspartate Transcarbamoylase Research Articles

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.

Quantitative urea gradient gel electrophoresis for studies of dissociation and unfolding of oligomeric proteins

Urea gradient gel electrophoresis combined with quantitative image processing of stained gels was used to analyze the dissociation and unfolding of the catalytic subunit of aspartate transcarbamoylase. The subunit, composed of three identical polypeptide chains, dissociates reversibly at high urea concentrations into unfolded chains. A comparison of the complex, but reproducible, gel patterns obtained for the native subunit and for the denatured protein in 6 M urea revealed significant differences at intermediate urea concentrations due to the presence of a transient kinetic intermediate identified as a relatively compact monomer. Mass transport equations based on a three state model were used to describe the urea gradient gel electrophoresis experiments, and a numerical solution yielded estimates of the population of molecular species and kinetic constants for the unfolding and refolding reactions as well as the dissociation and reconstitution reactions.

Association of the catalytic subunit of aspartate transcarbamoylase with a zinc‐containing polypeptide fragment of the regulatory chain leads to increases in thermal stability

The regulatory enzyme aspartate transcarbamoylase (ATCase), comprising 2 catalytic (C) trimers and 3 regulatory (R) dimers, owes its stability to the manifold interchain interactions among the 12 polypeptide chains. With the availability of a recombinant 70-amino acid zinc-containing polypeptide fragment of the regulatory chain of ATCase, it has become possible to analyze directly the interaction between catalytic and regulatory chains in a complex of simpler structure independent of other interactions such as those between the 2 C trimers, which also contribute to the stability of the holoenzyme. Also, the effect of the interaction between the polypeptide, termed the zinc domain, and the C trimer on the thermal stability and other properties can be measured directly. Differential scanning microcalorimetry experiments demonstrated that the binding of the zinc domain to the C trimer leads to a complex of markedly increased thermal stability. This was shown with a series of mutant forms of the C trimer, which themselves varied greatly in their temperature of denaturation due to single amino acid replacements. With some C trimers, for which tm varied over a range of 30 degrees C due to diverse amino acid substitutions, the elevation of tm resulting from the interaction with the zinc domain was as large as 18 degrees C. The values of tm for a variety of complexes of mutant C trimers and the wild-type zinc domain were similar to those observed when the holoenzymes containing the mutant C trimers were subjected to heat denaturation.(ABSTRACT TRUNCATED AT 250 WORDS)

Peptide-protein interaction markedly alters the functional properties of the catalytic subunit of aspartate transcarbamoylase.

Interaction of a 70-amino acid zinc-binding polypeptide from the regulatory chain of aspartate transcarbamoylase (ATCase) with the catalytic (C) subunit leads to dramatic changes in enzyme activity and affinity for ligand binding at the active sites. The complex between the polypeptide (zinc domain) and wild-type C trimer exhibits hyperbolic kinetics in contrast to the sigmoidal kinetics observed with the intact holoenzyme. Moreover, the Scatchard plot for binding N-(phosphonacetyl)-L-aspartate (PALA) to the complex is linear with a Kd corresponding to that evaluated for the holoenzyme converted to the relaxed (R) state. Additional evidence that the binding of the zinc domain to the C trimer converts it to the R state was attained with a mutant form of ATCase in which Lys 164 in the catalytic chain is replaced by Glu. As shown previously (Newell, J.O. & Schachman, H.K., 1990, Biophys. Chem. 37, 183-196), this mutant holoenzyme, which exists in the R conformation even in the absence of active site ligands, has a 50-fold greater affinity for PALA than the free C subunit. Adding the zinc domain to the C trimer containing the Lys 164-->Glu substitution leads to a 50-fold enhancement in the affinity for the bisubstrate analog yielding a value of Kd equal to that for the holoenzyme. A different mutant ATCase containing the Gln 231 to Ile replacement was shown (Peterson, C.B., Burman, D.L., & Schachman, H.K., 1992, Biochemistry 31, 8508-8515) to be much less active as a holoenzyme than as the free C trimer. For this mutant holoenzyme, the addition of substrates does not cause its conversion to the R state. However, the addition of the zinc domain to the Gln 231-->Ile C trimer leads to a marked increase in enzyme activity, and PALA binding data indicate that the complex resembles the R state of the holoenzyme. This interaction leading to a more active conformation serves as a model of intergenic complementation in which peptide binding to a protein causes a conformational correction at a site remote from the interacting surfaces resulting in activation of the protein. This linkage was also demonstrated by difference spectroscopy using a chromophore covalently bound at the active site, which served as a spectral probe for a local conformational change. The binding of ligands at the active sites was shown also to lead to a strengthening of the interaction between the zinc domain and the C trimer.

1H NMR studies on the catalytic subunit of aspartate transcarbamoylase.

The 1H NMR spectrum of the catalytic subunit of Escherichia coli aspartate transcarbamoylase (EC 2.1.3.2) was simplified by using strains auxotrophic for the aromatic amino acids and a growth medium containing fully deuterated Trp, Phe, and His and partially deuterated Tyr. 1H resonances for Tyr in the catalytic trimer (M(r) = 10(5)) were partially resolved into five peaks at 27 degrees C, which above 50 degrees C were further resolved to give a distinct resonance for each of the eight Tyr residues in the polypeptide chain. Experiments on chemically modified catalytic subunits and on a mutant form in which Tyr-165 was converted to Ser-165 led to the assignment of resonances for Tyr-165, Tyr-240, and Tyr-185. Binding of the substrate, carbamoyl phosphate, caused shifts of two of the unassigned resonances, and the subsequent binding of the aspartate analog succinate perturbed the resonances corresponding to Tyr-165 and Tyr-240. The bisubstrate analog N-(phosphonacetyl)-L-aspartate produced a spectrum differing considerably from that caused by the combination of carbamoyl phosphate and succinate. The NMR spectrum for the Tyr-165-->Ser mutant trimer showed clearly that the single amino acid substitution caused conformational changes affecting the environment of residues remote from the position of the replacement. In contrast, the inactive mutant subunit in which Gly-128 was replaced by Asp exhibited a spectrum virtually identical to that of the wild-type protein. However, addition of the substrate carbamoyl phosphate caused a marked change in the spectrum of the mutant enzyme, whereas that of the wild-type trimer was altered only slightly, showing that the effect of the amino acid substitution was manifested in the NMR spectrum only with the liganded enzyme.

Study of strong to ultratight protein interactions using differential scanning calorimetry

Data from differential scanning calorimetry (DSC) may be used to estimate very large binding constants that cannot be conveniently measured by more conventional equilibrium techniques. Thermodynamic models have been formulated to describe interacting systems that involve either one thermal transition (protein-ligand) or two thermal transitions (protein-protein) and either 1:1 or higher binding stoichiometry. Methods are described for obtaining binding constants and heats of binding by two different methods: calculation or simulation fitting of data. Extensive DSC data on 2'CMP binding to RNase are presented and analyzed by the two methods. It is found that the methods agree when binding sites are completely saturated, but substantial errors arise in the calculation method when site saturation is incomplete and the transition of liganded molecules overlaps that of unliganded molecules. This arises primarily from an inability to determine TM (i.e., the temperature where concentrations of folded and unfolded protein are equal) under weak-binding conditions. Results from simulation show that the binding constants and heats of binding from the DSC method agree quantitatively with corresponding estimates obtained from equilibrium methods when extrapolated to the same temperature. It was also found from the DSC data that the binding constant decreases with increasing concentration of ligand, which might arise from nonideality effects associated with dimerization of 2'CMP. Simulations show that the DSC method is capable of estimating binding constants for ultratight interactions up to perhaps 10(40) M-1 or higher, while most equilibrium methods fail well below 10(10) M-1. DSC data from the literature on a number of interacting systems (trypsin-soybean trypsin inhibitor, trypsin-ovomucoid, trypsin-pancreatic trypsin inhibitor, chymotrypsin-subtilisin inhibitor, subtilisin BPN-subtilisin inhibitor, RNase S protein-RNase S peptide, avidin-biotin, ovotransferrin-Fe3+, superoxide dismutase-Zn2+, alkaline phosphatase-Zn2+, and assembly of regulatory and catalytic subunits of aspartate transcarbamoylase) were analyzed by simulation fitting or by calculation. Apparent single-site binding constants ranged from ca. 10(5) to 10(20) M-1, while the interaction constant for assembly of aspartate transcarbamoylase was estimated as 10(37) in molarity units. For most of these systems, the DSC interaction constants compared favorably with other literature estimates, for some it did not for reasons unknown, while for still others this represented the first estimate. Simulations show that for proteins having two binding sites for the same ligand within a single cooperative unit, ligand rearrangement will occur spontaneously during a DSC scan as the transition temperature of the unliganded protein is approached.(ABSTRACT TRUNCATED AT 400 WORDS)

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].

Shared active sites in oligomeric enzymes: model studies with defective mutants of aspartate transcarbamoylase produced by site-directed mutagenesis.

Many oligomeric enzymes are functional only in the assembled form, and it is often difficult to determine unambiguously why monomers are inactive. In some cases individual monomers cannot fold into stable correct ("native") conformations without contributions from interchain interactions. For other oligomers, catalysis requires the contributions of amino acid residues at the interface between adjacent polypeptide chains, and monomers are inactive because they cannot form complete active sites. A test for the presence of shared sites was devised that is based on the formation of active hybrid oligomers from appropriate inactive parental mutants produced by site-directed mutagenesis. This approach was applied in a study of the catalytic trimer of aspartate transcarbamoylase (aspartate carbamoyltransferase, EC 2.1.3.2) from Escherichia coli, using three mutants, in which Ser-52 was replaced by His, Lys-84 by Gln, or His-134 by Ala. Hybrid trimers formed from the virtually inactive Ser and Lys mutants were 10(5) more active than the parental proteins, and the specific activities of each hybrid were about 33% that of the wild-type trimer, as expected for the scheme based on shared sites. Hybrids from the His and Lys mutants had comparable specific activities. Moreover, one hybrid with approximately 33% activity had one high-affinity binding site for a bisubstrate analog as compared to about three for wild-type trimer. As a further test, hybrids were also formed from wild-type and double-mutant (Lys-84----Gln and His-134----Ala) trimers. The hybrid containing two chains with the double mutation and one wild-type chain had very little activity, and that composed of one double mutant and two wild-type chains had 32% the specific activity of wild-type trimers. This negative complementation experiment is in quantitative accord with the scheme based on shared sites at or near the interfaces between adjacent chains. The techniques used to demonstrate shared active sites in the catalytic subunits of aspartate transcarbamoylase can be applied generally to various types of oligomers (dimers, tetramers, etc.) to determine whether the participation of amino acid residues from adjoining chains is essential for forming active sites in oligomeric enzymes.

Kinetics of the interaction of N-(phosphonacetyl)-L-aspartate with the catalytic subunit of aspartate transcarbamoylase. A slow conformational change subsequent to binding.

The binding of the bisubstrate ligand N-(phosphonacetyl)-L-aspartate (PALA) to the active sites of both the free catalytic subunit of aspartate transcarbamoylase and the intact holoenzyme causes conformational changes which have been studied extensively. However, no kinetic information has been available about the sequence of events occurring during the formation or dissociation of the complexes. Stopped flow kinetics, 31P saturation transfer NMR spectroscopy, and presteady-state kinetics were used to monitor the interaction of PALA with the catalytic subunit (or a derivative containing nitrotyrosyl chromophores which served as spectral probes). The various experimental approaches lead to a mechanism that includes a rapid binding of PALA with an "on" rate of about 10(8)M-1s-1 and an "off" rate of 28 s-1, followed by a much slower isomerization of the complex with a forward rate constant of 0.18 s-1. Analysis of the presteady-state bursts of enzyme activity when the protein is added to a mixture of substrates and PALA and of the lag in activity when the PALA complex with catalytic subunit is added to substrates yielded a rate constant for the reverse isomerization of 0.018s-1. Thus, the conformational change subsequent to PALA binding leads to a 10-fold increase in the equilibrium constant for complex formation. Stopped flow kinetic measurements of the spectral change resulting from mixing the complex of PALA and nitrated protein with native enzyme showed a slow process with a t1/2 of about 11 s, whereas 31P saturation transfer NMR experiments yielded at t1/2 of about 260 ms for the dissociation of PALA from the complex. This apparent disparity is understood in terms of the two-step binding scheme where rapid dissociation of the initial ligand X enzyme complex is measured by the NMR technique and the slow isomerization of the complex is responsible for the bulk of the stopped flow signal.

The influence of quaternary structure on the active site of an oligomeric enzyme. Catalytic subunit of aspartate transcarbamoylase.

The catalytic subunit of aspartate transcarbamoylase from Escherichia coli reacts readily with 2,4,6-trinitrobenzenesulfonate, resulting in the loss of enzymatic activity. Substrates and substrate analogs protect the enzyme in a competitive manner, indicating that the loss of activity is due to modification of active-site residues. This conclusion was confirmed by fractionating tryptic digests of the modified protein followed by the identification of active-site lysines 83 and 84 as the modified residues. When three trinitrophenyl groups are incorporated per catalytic trimer, 70% of the activity is lost. The modified protein retains the sedimentation velocity and electrophoretic properties of the native catalytic subunit and can associate with regulatory subunit to form a holoenzyme-like molecule. The trinitrophenylated catalytic trimers have two strong absorption bands at 345 and 420 nm which serve as sensitive spectral probes in difference-spectroscopy experiments. Results from such experiments show that 1) the modified trimeric enzyme binds active-site ligands; 2) dissociation of the trimer into compact, highly structured monomers gives a spectral response distinguishable from that observed when the chains are completely unfolded; and 3) even though dissociation of the trimers to folded monomers causes the complete loss of enzyme activity, the resulting monomers still retain the ability to bind the bisubstrate analog N-(phosphonacetyl)-L-aspartate. These results indicate that the active site must be at least partially formed in the absence of any quaternary structure.

Amino acid sequence of the catalytic subunit of aspartate transcarbamoylase from Escherichia coli.

We propose a primary structure for the catalytic subunit of aspartate transcarbamoylase (aspartate carbamoyltransferase; carbamoylphosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2) from Escherichia coli based on amino acid sequences of fragments obtained by cyanogen bromide cleavage, by tryptic digestion of the succinylated polypeptide, and by chymotryptic and proteinase C digestion of the intact catalytic chain. The protein contains 310 amino acids and has a calculated molecular weight of 33,944. The negatively and positively charged residues are distributed uniformly, and there is no indication of charge clustering in the linear sequence.

Nucleotide sequence of the structural gene (pyrB) that encodes the catalytic polypeptide of aspartate transcarbamoylase of Escherichia coli.

The deoxyribonucleotide sequence of pyrB, the cistron encoding the catalytic subunit of aspartate transcarbamoylase (carbamoylphosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2), has been determined. The pyrB gene encodes a polypeptide of 311 amino acid residues initiated by an NH2-terminal methionine that is not present in the catalytically active polypeptide. The DNA sequence analysis revealed the presence of an eight-amino-acid sequence beginning at Met-219 that was not detected in previous analyses of amino acid sequence. This octapeptide sequence provides an additional component of the disordered loop in the equatorial domain of the catalytic polypeptide. It had been found previously that the catalytic polypeptide is expressed from a bicistronic operon that also produces the regulatory polypeptide encoded by pyrI. A single transcriptional control region precedes the structural gene of the catalytic polypeptide and a simple 15-base-pair region separates its COOH terminus from the structural gene of the regulatory polypeptide. The chain-terminating codon of the catalytic polypeptide may contribute to the ribosomal binding site for the regulatory polypeptide and thus assist coordinate expression of the two cistrons.

Ligand-promoted strengthening of interchain bonding domains in catalytic subunits of aspartate transcarbamoylase.

Ligand-promoted strengthening of interchain bonding domains in catalytic subunits of aspartate transcarbamoylase.

Assembly of the catalytic trimers of aspartate transcarbamoylase from unfolded polypeptide chains.

In an effort to stimulate the in vivo formation of active enzyme from newly synthesized polypeptide chains, we have studied the in vitro assembly of the active catalytic subunits of aspartate transcarbamoylase from unfolded polypeptide chains. Hydrodynamic and spectroscopic measurements showed that incubating the catalytic trimers in 4.7 M urea for 45 min at 9 degrees C produced unfolded polypeptide chains largely devoid of the secondary and tertiary structures characteristic of native subunits. Dilution of the urea solutions led to the slow restoration of enzyme activity and the formation of trimers at a rate which could be measured quantitatively by a hybridization technique using succinylated polypeptide chains as a "chase" to "stop" the assembly. Kinetic studies showed that reactivation and assembly of trimers were coincident with a half-time for completion of about 50 min at 0 degrees C. Also, the rate-limiting reaction was first order. Although these results suggest that chain folding is the slow process, spectroscopic studies indicated that large changes in the environments of the aromatic amino acid residues occur very rapidly. Indeed the changes in the absorption spectrum are largely complete before significant reactivation and trimer formation occur. The results are consistent with an assembly mechanism in which the first step is the rapid collapse of the expanded randomly coiled chains to give partially folded monomers. These monomers are not enzymically active and cannot associate to form trimers until a rate-limiting conformational change occurs. Subsequent to this slow process, the "competent" monomers associate via a series of reactions to form active trimers.

Assembly of catalytic subunits of aspartate transcarbamoylase from Escherichia coli

Although extensive studies have been conducted on the assembly of the allosteric enzyme, aspartate transcarbamoylase (ATCase) from isolate, intact catalytic (C) and regulatory (R) subunits, there has been little research on the formation of these subunits from individual catalytic (c) and regulatory (r) polypeptide chains. Such studies would be useful for evaluating the strengths of the interchain bonding domains within the subunits just as earlier experiments provided valuable data regarding interactions between the subunits in ATCase. The intact enzyme comprising two C trimers and three R dimers is designated as C/sub 2/R/sub 3/ or c/sub 6/r/sub 6/.

Heterogeneity of sites in isolated catalytic subunits of aspartate transcarbamoylase.

Carbamoyl phosphate, a substrate of aspartate transcarbamoylase from Escherichia coli, binds with different modes of association in 3 sites in the unmodified catalytic subunits. Over a narrow pH range (6.6--8.0), positive, negative or no interactions are observed. Several substrate analogues also bind to 3 sites in the catalytic trimer. The association of pyridoxal phosphate, CTP and ATP, all competitive inhibitors of carbamoyl phosphate, exhibit negative interactions. Binding of succinate, an analogue of the second substrate, aspartate, is also characterized by heterogeneity. Dissociation constants to high and low-affinity sites differ by factors of 10-100. These observations clearly indicate that, although not observed kinetically, the active sites in the catalytic subunits of aspartate transcarbamoylase are heterogenous.