Catalytic Subunit Of Aspartate Transcarbamylase Research Articles

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.

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.

Construction of a cDNA to the hamster CAD gene and its application toward defining the domain for aspartate transcarbamylase.

cDNA complementary to hamster mRNA encoding the CAD protein, a multifunctional protein which carries the first three enzymes of pyrimidine biosynthesis, was constructed. The longest of these recombinants (pCAD142) covers 82% of the 7.9-kilobase mRNA. Portions of the cDNA were excised and replaced by a lac promoter-operator-initiation codon segment. The resultant plasmids were transfected into an Escherichia coli mutant defective in aspartate transcarbamylase, the second enzyme of the pathway. Complementation of the bacterial defect was observed with as little as 2.2 kilobases of cDNA sequence, corresponding to the 3' region of the mRNA. DNA sequencing in this region of the hamster cDNA reveals stretches which are highly homologous to the E. coli gene for the catalytic subunit of aspartate transcarbamylase; other stretches show no homology. The highly conserved regions probably reflect areas of protein structure critical to catalysis, while the nonconserved regions may reflect differences between the quaternary structures of E. coli and mammalian aspartate transcarbamylases, one such difference being that the bacterial enzyme in its native form is allosterically regulated and the mammalian enzyme is not.

Modification of three active site lysine residues in the catalytic subunit of aspartate transcarbamylase by D- and L-bromosuccinate.

Treatment of the catalytic subunit of aspartate transcarbamylase from Escherichia coli with either D- or Lbromosuccinate at pH 8.5 results in a loss of catalytic activity. Succinate, an analog of the substrate L-aspartate, affords some protection against inactivation, while the putative transition state analogN-(phosphonacetyl)-L-aspartate provides complete protection. The substrate carbamyl phosphate provides greater protection against inactivation by L-bromosuccinate than by D-bromosuccinate. Complete loss of activity is accompanied by incorporation of approximately 1.3 succinate moieties per catalytic chain resulting from partial modification of 3 lysine residues, identified as numbers 83, 84, and 224 in the preliminary catalytic chain sequence of W. Konigsberg (personal communication). A significant number of catalytic chains are modified at both positions 83 and 84. In the absence of ligands, D-bromosuccinate reacts with lysine 83 to a greater extent than does the L isomer. Bulky inhibitors such as CTP and pyridoxal 5’-phosphate provide varying degrees of protection against inactivation and overall modification without altering significantly the relative extent of alkylation of the 3 residues. However, carbamyl phosphate not only protects against inactivation and overall modification, but also selectively suppresses alkylation of lysine 83 and eliminates the production of catalytic chains modified at both lysines 83 and 84. The results suggest that all 3 lysine residues are at or near the active site, that modification of any one of them causes loss of catalytic activity, and that lysine 83 is at or near the carbamyl phosphate binding site.

Comparison of the essential arginine residue in Escherichia coli ornithine and aspartate transcarbamylases

The reaction of phenylglyoxal with Escherichia coli ornithine transcarbamylase (carbamoylphosphate: l-ornithine carbamoyltransferase, EC 2.1.3.3) leads to complete loss of enzymatic activity. The behavior of this reagent towards ornithine transcarbamylase is remarkably similar to that observed with E. coli aspartate transcarbamylase (carbamoylphosphate: l-aspartate carbamoyltransferase, EC 2.1.3.2) and its catalytic subunit (Kantrowitz, E.R. and Lipscomb, W.N. (1976) J. Biol. Chem. 251, 2688–2695). The rate of phenylglyoxal inactivation increases in the order ornithine transcarbamylase, catalytic subunit of aspartate transcarbamylase and aspartate transcarbamylase. For ornithine transcarbamylase, the substrate carbamyl phosphate alone or in combination with the substrate analog norvaline protect the enzyme from phenylglyoxal inactivation. Under similar conditions, carbamyl phosphate or carbamyl phosphate plus succinate will protect the catalytic subunit of aspartate transcarbamylase in an almost identical manner. Using [ 14C]phenylglyoxal, the number of arginine residues involved in loss of activity was determined to be approx. three per ornithine transcarbamylase molecule or one arginine per active site. The data suggest that the arginine necessary for activity is involved in the binding of carbamyl phosphate to the enzyme. The similarity in phenylglyoxal reactivities combined with genetic and structural data suggest very strongly that there is an evolutionary relationship between ornithine transcarbamylase and the catalytic subunit of aspartate transcarbamylase.

Crystallization and preliminary X-ray study of the catalytic subunit of aspartate transcarbamylase

Crystals of the catalytic subunit of aspartate transcarbamylase have been obtained from 30 to 40% ammonium sulphate at pH 8. The crystals are tetragonal with space group P4 12 12 (or P4 32 12) and unit cell dimensions a = b = 82 A ̊ , c = 298 A ̊ , and contain one trimer per asymmetric unit. They diffract to about 3.5 Å.

Inactivation of the catalytic subunit of aspartate transcarbamylase by nitration with tetranitromethane.

Inactivation of the catalytic subunit of aspartate transcarbamylase by nitration with tetranitromethane.

Specifically deuterated l-malate as an NMR probe of the aspartate transcarbamylase catalytic site

Replacement of protons by deuterium at specific positions of l-malate results in simplified proton magnetic resonance spectra and a large reduction in the effect of intramolecular dipole-dipole relaxation of the proton resonances. Under conditions of concomitant deuterium decoupling, these spectra are further simplified. These advantages are used to study the proton relaxation of two specifically deuterated l-malates in the presence of the catalytic subunit of aspartate transcarbamylase. The bound relaxation rates of the deuterated L-malates are much smaller than those of undeuterated l-malate implying that the major relaxation mechanism for enzyme bound, undeuterated l-malate is the intramolecular dipole-dipole interaction.

Aspartate Transcarbamylase: A STUDY OF POSSIBLE ROLES FOR THE SULFHYDRYL GROUP AT THE ACTIVE SITE

Abstract Potential roles for the single sulfhydryl group of the catalytic subunit of aspartate transcarbamylase from Escherichia coli have been investigated by means of chemical modification. The S-cyano and S-methyl derivatives are highly active and recombine normally with the zinc regulatory subunit. The derivatives of native enzyme formed in this way are fully responsive to homotropic and heterotropic allosteric effectors and have pH-activity curves indistinguishable from that of unmodified native enzyme. Therefore, despite strong evidence that the—SH group is at the active site, its integrity is not necessary for activity or for allosteric function. However, modifications which lead to negatively charged derivatives of the—SH group do result in inactivation. The reason for such loss of activity was investigated with a sulfonate derivative prepared by rapid and highly specific oxidation of the native enzyme with KMnO4. Carbamyl phosphate and the transition state analog N-(phosphonacetyl)-l-aspartate bind well to the sulfonate derivative, but the affinity of this derivative for succinate (in the presence of carbamyl-P) is much reduced when compared to that of native enzyme. The newly introduced negative charge apparently inactivates by greatly reducing the affinity of the active site for the substrate l-aspartate. The trimeric catalytic subunit is inactive after modification of the 3 identical —SH groups by dithionitrobenzoate, and the derivative is capable of binding tightly only 1 eq of either carbamyl-P or N-(phosphonacetyl)-l-aspartate per trimer, demonstrating a strong interaction among the three active sites of the isolated subunit. Furthermore, the thionitrobenzoate derivative of native aspartate transcarbamylase correspondingly binds only about 2 molecules of N-(phosphonacetyl)-l-aspartate per six active sites and binds them with strong positive cooperativity, suggesting that in unmodified native enzyme positive cooperativity is due at least in part to an interaction between the two catalytic trimers. If catalytic subunit is combined with a limiting amount of zinc regulatory subunit, a new species deficient in regulatory subunit is formed, with altered allosteric properties.

Conformational studies on the nitrated catalytic subunit of aspartate transcarbamylase

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTConformational studies on the nitrated catalytic subunit of aspartate transcarbamylaseMarc W. Kirschner and H. K. SchachmanCite this: Biochemistry 1973, 12, 16, 2987–2997Publication Date (Print):July 1, 1973Publication History Published online1 May 2002Published inissue 1 July 1973https://doi.org/10.1021/bi00740a007RIGHTS & PERMISSIONSArticle Views52Altmetric-Citations26LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (2 MB) Get e-Alerts Get e-Alerts

Interaction between polypeptide chains within the catalytic subunit of aspartate transcarbamylase

The catalytic subunit of aspartate transcarbamylase, which normally exhibits Michaelis-Menten kinetics, gives rise to nonlinear double reciprocal plots of initial velocity data in the presence of the inhibitor ITP. The results of kinetic and binding studies on the enzyme in the presence of ITP are considered in conjunction with the fact that the catalytic subunit is composed of three polypeptide chains. The data are consistent wih the proposition that the combination of carbamyl phosphate on one polypeptide chain can hinder the combination of ITP on other polypeptide chains, and vice versa. Thus the isolated catalytic subunit is capable of exhibiting a type of cooperative effect, although this effect is only observed in the presence of the inhibitor.

Difficulties encountered with an ultrafiltration method for measuring the binding of ligands to protein

The ultrafiltration method of Paulus has been used to study the binding of aspartate and ITP to the catalytic subunit of aspartate transcarbamylase. Markedly different estimates of dissociation constants and of the number of moles of ligand bound per mole of enzyme have been obtained using different batches of UM 10 Diaflo membranes. Estimates derived using Visking membranes are presented for comparison. For both an amino acid and a nucleotide invalid results have been obtained using UM 10 Diaflo membranes in conjunction with this enzyme. The importance of excluding artifacts in any system studied by this method is emphasized.

Conformational changes in proteins as measured by difference sedimentation studies. II. Effect of stereospecific ligands on the catalytic subunit of aspartate transcarbamylase.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTConformational changes in proteins as measured by difference sedimentation studies. II. Effect of stereospecific ligands on the catalytic subunit of aspartate transcarbamylaseMarc W. Kirschner and H. K. SchachmanCite this: Biochemistry 1971, 10, 10, 1919–1926Publication Date (Print):May 11, 1971Publication History Published online1 May 2002Published inissue 11 May 1971https://doi.org/10.1021/bi00786a028RIGHTS & PERMISSIONSArticle Views73Altmetric-Citations64LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (3 MB) Get e-Alerts Get e-Alerts

Aspartate Transcarbamylase: A Nuclear magnetic resonance study of the binding of inhibitors and substrates to the catalytic subunit

Abstract The binding of small molecules to macromolecules can be studied by nuclear magnetic resonance since the width of a resonance can often be related to rotational correlation times and exchange rates for bound and unbound species. Line widths for carbon-linked protons of analogues of carbamyl phosphate (carbamyl-P) in the presence of the catalytic subunit of aspartate transcarbamylase are a measure of the rotational freedom of the bound analogues. The protons of phosphonacetamide are broadened considerably in the presence of the subunit, while those of N-methyl phosphonacetamide and methyl phosphonate are not, indicating that an interaction between the [see PDF for structure] group of the analogue and the enzyme limits rotation about the P—C bond in the first instance but not in the latter two. The resonance line width for the methylene protons of 0.025 m succinate, a competitive inhibitor of aspartate, is 0.48 Hz in the absence of enzyme at pH 7, increases slightly when catalytic subunit (20 mg per ml) is added, but increases to 2.00 Hz upon the further addition of 0.025 m carbamyl-P. In this case, the broadening is due primarily to an increase in the lifetime of the succinate-enzyme complex, induced by carbamyl-P. Some analogues of carbamyl-P, for example phosphonacetamide and acetyl phosphate, induce broadening but others, including N-methyl phosphonacetamide, N-methyl carbamyl phosphate, and methyl phosphonate, do not. Only analogues which are not larger than carbamyl-P and which have a carbonyl group in addition to a phosphate or phosphonate dianion induce the broadening of the succinate line. The resonance for the protons of malonate, another inhibitor of aspartate, is also broadened by carbamyl-P and phosphonacetamide in the presence of the catalytic subunit. Dissociation constants determined at pH 7 confirm that carbamyl-P is the most effective analogue in inducing succinate binding at this pH. In addition to line widths, chemical shifts of the bound species have been determined for some of the analogues. The chemical shifts suggest that an aromatic ring is near the binding site for phosphate.

Aspartate Transcarbamylase: Studies of the catalytic subunit by ultraviolet difference spectroscopy

Abstract The binding of substrates and substrate analogues to the catalytic subunit of aspartate transcarbamylase from Escherichia coli can be detected by difference spectroscopy between 275 and 310 mµ; the chromophores giving rise to these difference spectra are tentatively identified as a tryptophyl residue perturbed by the binding of carbamyl phosphate and a tyrosyl residue perturbed by the binding of l-aspartate. Dissociation constants for several l-aspartate analogues have been determined by difference spectroscopy. When the carbamyl phosphate site is unoccupied, l-aspartate, succinate, l-malate, and d-malate each bind weakly at the l-aspartate site. In the presence of carbamyl phosphate, only succinate and d-malate, the two dicarboxylic acids which lack l-α-substituents, bind tightly to the enzyme. In striking contrast, when carbamyl phosphate is replaced by phosphate, only l-aspartate and l-malate, the two dicarboxylic acids which have l-α-substituents, bind tightly to the enzyme. The magnitudes of the difference spectra for the ternary complexes are substantially greater than the sum of the magnitudes of the binary complexes formed by each analogue individually only when binding of the dicarboxylic acid is enhanced. We suggest, as an explanation for these observations, that the enzyme converts a portion of the electrostatic binding energy of l-aspartate into energy for the mechanical act of forcing the substrates together. A detailed model for the catalytic mechanism is presented, based on the data presented in this and the accompanying papers.

Aspartate Transcarbamylase: Kinetic studies of the catalytic subunit

Abstract 1. Steady state kinetics indicates that the binding of substrates by the catalytic subunit of aspartate transcarbamylase is ordered. Product inhibition patterns show that carbamyl phosphate binds first, aspartate binds second, carbamylaspartate dissociates first, and phosphate dissociates second. 2. Nonlinear product inhibition and substrate inhibition indicate that several dead end complexes are formed: aspartate binds to the enzyme-phosphate complex, a 2nd mole of phosphate binds to the enzyme-phosphate complex, a 2nd mole of carbamyl phosphate binds to the enzyme-carbamyl phosphate complex, and carbamylaspartate binds weakly to the free enzyme. 3. The interaction of the catalytic subunit with analogues of both substrates has been studied in an attempt to reveal those structural features of the substrates that are required for binding and function. Acetyl phosphate and N-methylcarbamyl phosphate are the only analogues of carbamyl phosphate tested which are substrates, with maximum velocities at pH 7.8 of 2.4% and 0.03% of the maximum velocity with carbamyl phosphate. Since acetyl phosphate lacks the NH2 group of carbamyl phosphate, this group cannot be essential for function. N,N-Dimethylcarbamyl phosphate is not a substrate, but it is a competitive inhibitor, as is any analogue of carbamyl phosphate with a phosphate or phosphonate dianion, suggesting that at least part of the site for carbamyl phosphate is readily accessible. In fact, with the catalytic subunit, even cytidine triphosphate is a competitive inhibitor. 4. Several dicarboxylic acids were found to be competitive inhibitors of l-aspartate. Succinate and maleate are the strongest inhibitors, malonate and d- and l-malate are good inhibitors, whereas fumarate, glutarate, and d- and l-bromosuccinate are poor inhibitors, and d-aspartate does not inhibit significantly. 5. The pH dependence of the dissociation constant for succinate indicates that a group with pKa 7.1 is required to be positively charged for this inhibitor to bind to the enzyme carbamyl phosphate complex. In contrast, the dissociation constants for carbamyl phosphate and phosphonacetamide vary little between pH 6 and pH 9. 6. Product inhibition by carbamylaspartate when aspartate is varied indicates that the constant for dissociation of aspartate from the central complex is much larger than the dissociation constants for unreactive aspartate analogues such as succinate. This indicates that some of the binding energy of aspartate may be used to facilitate the reaction with carbamyl phosphate. 7. The catalytic subunit is unstable at concentrations below 1 µg per ml. There is an immediate decrease in specific activity with decreasing enzyme concentration and a slower irreversible inactivation during incubation at low concentrations. Both the immediate and the slow losses of activity are prevented by the presence of bovine serum albumin (50 µg per ml) in dilute enzyme solutions. However, because bovine serum albumin forms a complex with the catalytic subunit and alters the apparent kinetic parameters, it was not used in these kinetic studies. 8. At pH 7.8 and 28°, the dissociation constant for carbamyl phosphate is 1.4 x 10-5 m, the Km for aspartate is 0.020 m, and the Vmax is 9.0 x 104 moles per min per 100,000 g of subunit. The dissociation constant for carbamyl phosphate and Km for aspartate vary little between pH 6.4 and pH 8.7, whereas Vmax changes greatly, with an optimum near pH 8. With the use of acetyl phosphate instead of carbamyl phosphate, Km for aspartate is unchanged at pH 7.8, but the dissociation constant for acetyl phosphate is 35-fold higher. With N-methylcarbamyl phosphate, Km for aspartate is 7-fold higher and the dissociation constant for N-methylcarbamyl phosphate is 4-fold higher than for carbamyl phosphate.