Allosteric Interactions in Aspartate Transcarbamylase. II. Evidence for Different Conformational States of the Protein in the Presence and Absence of Specific Ligands

Gerhart, John C.; Schachman, H. K.

doi:10.1021/bi00842a600

Cited by 324 publications

(212 citation statements)

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“…5 , 6 ; Table I (Schachman, 1988). Direct evidence for this change in quaternary structure was first revealed for the wild-type enzyme by the 3% decrease in the sedimentation coefficient accompanying the ligand-promoted T to R transition (Gerhart & Schachman, 1968). As seen in Table 1, the addition of the bisubstrate analogue PALA leads to a decrease in sedimentation coefficient (Table l) for cpATCasec,22, cpATCa~ec22Z, and ,pATcase,281, which is very similar to that observed for the wild-type enzyme under comparable conditions ( A s h is about -2.5%).…”

Section: Discussionmentioning

confidence: 99%

“…As seen in Table 1, all of the variants showed a decrease in the sedimentation coefficient of about 2.5%, in excellent agreement with the value observed for the wild-type enzyme under similar conditions. Such a decrease in sedimentation coefficient corresponds to a ligand-promoted conformational change from a compact structure to a more swollen form (Gerhart & Schachman, 1968;Howlett & Schachman, 1977).…”

Section: Catalytic and Regulatory Properties Of Atcase Variants Contamentioning

confidence: 99%

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

Zhang

Schachman

1996

Protein Science

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Because the N-and C-terminal amino acids of the catalytic (c) polypeptide chains of Escherichia coliaspartate 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 V,, for cpATCase122, cpATCase222, and , , A T C~S~~,~ ranged from 16-21 pmol carbamoylaspartate per pg per h, compared with 15 for wild-type ATCase, and the values for 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 for aspartate and a decrease in the Hill coefficient (nH). In contrast, CTP caused both an increase in 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 ~-(phosphonacety1)-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.

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Section: Discussionmentioning

confidence: 99%

Section: Catalytic and Regulatory Properties Of Atcase Variants Contamentioning

confidence: 99%

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

Zhang

Schachman

1996

Protein Science

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“…One can argue from these data that the symmetry of this enzyme when bound to molecules of this substrate analogue is D3 from the former and at least C3 from the latter crystals. If, as sedimentation ultracentrifuge experiments suggest (28), there is a large conformational change, it probably is at least an elongation of the molecule along the threefold axis, a change that is consistent with these unit cell measurements but that is not proved by them. …”

mentioning

confidence: 80%

Three-dimensional structures of aspartate carbamoyltransferase from Escherichia coli and of its complex with cytidine triphosphate.

Monaco¹,

Crawford²,

Lipscomb³

1978

Proc. Natl. Acad. Sci. U.S.A.

149

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X-ray diffraction studies to nominal resolutions of 3.0 A for unliganded aspartate carbamoyltransferase (EC 2.1.3.2) (R32 crystal symmetry) and of 2.8 A for the complex of aspartate carbamoyltransferase with cytidine triphosphate (P321 crystal symmetry) have yielded traces of the polypeptide chains of the catalytic (C) and regulatory (R) (1,2). In Escherichia coli this reaction is the first unique step in the biosynthesis of pyrimidines. The endproduct of this pathway, cytidine triphosphate (CTP) inhibits this allosteric enzyme by interacting with a regulatory chain R, which is distinct from the catalytic chain C (3, 4). Also ATP, a purine, stimulates this enzyme so that a tendency toward balance in the pyrimidine and purine syntheses is achieved. The enzyme is hexameric (5, 6), and can be dissociated by use of mercurials into two catalytic trimers C3 and three regulatory dimers R2 (7). Molecular weights are 34,000 for C and 17,000 for R (5,8,9). Each regulatory unit contains one Zn2 , which has been presumed to coordinate to the four sulfhydryl groups of this chain (10, 11), and which is necessary for reconstitution of the enzyme from the subunits C3 and R2. Other properties of this enzyme have been summarized recently (12).Two crystallographic forms of molecular aspartate carbamoyltransferase have been studied at low resolution in this laboratory. Crystals of R32 symmetry of the unliganded form (containing neither allosteric effectors nor substrate analogues) show at 5.5-A resolution that the molecular symmetry is D3, that the two catalytic trimers are almost eclipsed when viewed along the molecular threefold axis, and that there is a large aqueous cavity in the center of the molecule (13). Crystals of symmetry P321 of the form grown in the presence of CTP showed that there are no gross structural differences when compared to the unliganded R32 form (14). The additional electron density associated with CTP was verified by a further differencedensity study in which 5-iodo-CTP was bound to the enzyme. In this brief paper we summarize x-ray diffraction studies at 3.0-A resolution for the unliganded R32 form and at 2.8-A resolution for the CTP-P321 form. The major features of secondary structure are presented below.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 5276 STRUCTURE DETERMINATION The unliganded R32 form has one RC group per asymmetric unit in a unit cell of dimensions a = 132 A and c = 199 A, while the CTP-P321 form has two RC units per crystallographic asymmetric unit in a unit cell of dimensions a = 122 A and c = 142 A. The x-ray diffraction data were collected by use of the oscillation method (15) from the native enzyme, and from heavy atom derivatives listed in Table 1. Conditions of preparation of these derivatives have been given previously (13,14), except for the third CTP-P321 derivative, for which crystals were immersed ...

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“…However, conventionally prepared R subunit neither contains zinc ions, nor does it take up stoichiometric amounts of Zn or Cd without prior denaturation. It has optical properties (2,7,11) Both mechanisms can also explain the greatly increased reactivity of the sulfhydryl groups in the isolated R subunit. The reactivity of the metal ions and of the sulfhydryl groups closely parallel each other in that they exhibit high reactivity in the R subunit and low reactivity in the full enzyme.…”

Section: Discussionmentioning

confidence: 99%

Localization of the Zinc Binding Site of Aspartate Transcarbamoylase in the Regulatory Subunit

Rosenbusch

Weber

1971

Proc. Natl. Acad. Sci. U.S.A.

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Aspartate transcarbamoylase (EC 2.1.3.2) from Escherichia coli contains six zinc ions per molecule. (1,4) data that the polypeptide chains are arranged around one three-fold and three two-fold axes of symmetry.* If all six metal ions are situated in spatially equivalent positions, they must be localized either in the catalytic or in the regulatory chains or at the subunit contacts. This report describes renaturation experiments that allow one to localize the binding sites in the regulatory subunits. MATERIALS AND METHODSAspartate transcarbamoylase was purified as described by Gerhart and Holoubek (5). Cadmium enzyme was prepared in vivo by substitution of ZnSO4 by CdCl2 in the growth medium. Purified Zn and Cd enzymes were interconverted in vitro by successive denaturation, replacement of the metal, and renaturation. Enzyme was denatured in 6 M guanidineHCl, 0.1 M 0-mercaptoethanol, and 1 mM EDTA. After 4 hr of incubation at 200C, the protein was subjected to gel filtration on Sephadex G-25 in 6 M urea-20 mM #-mercapto- (1) has been described in detail.Metal derivatives of the regulatory subunit were prepared as follows. 250 ml of enzyme solution [2 mg/ml in 4 mM potassium phosphate (pH 7.0)-0.4 mM EDTA-2 mM ,5-mercaptoethanol] was heated to 640C for 8 min, then rapidly cooled in ice and centrifuged. The supernatant contained 60-70% of the theoretical amount of C subunit, no R chains (as judged by acrylamide gel electrophoresis in sodium dodecyl sulfate), and Zn ions in quantitative amounts. The pellet consisted of R subunit and the remaining fraction of C subunits. All further experiments with isolated R subunit were performed in 0.2 M Tris acetate at pH 8.2 (buffer A). The pellet was dissolved in 4 ml. of buffer A-8 M guanidine -HCl-0.13 M j3-mercaptoethanol-1.3 mM EDTA. After a 4-hr incubation, gel filtration in urea was performed as described above. Protein fractions were pooled, and Zn or Cd was added in a 1.5-molar excess of the metal binding sites. R subunits were renatured by dripping the protein into eleven volumes of buffer A-0.1 M g-mercaptoethanol, followed by a 4-hr incubation at 4VC. After concentration by ultrafiltration, EDTA was added (1 mM), and the protein was subjected to gel filtration in buffer A-2 mM #-mercaptoethanol. R subunit was separated by zone centrifugation from a small amount of the complete enzyme. The total recovery was 50%.Apo R subunit was prepared by an identical procedure, except that no metal ions were added before renaturation.This derivative still contained about 20% of the original complement of zinc ions. R subunit, prepared by pMB cleavage of enzyme (5), was used as an alternative starting material to obtain metal derivatives: 5-10 mg/ml of this subunit in buffer A was made 0.1 M in j#-mercaptoethanol, and then 6 M in guanidine HCI. After gel filtration in 6 M buffered urea-20 mM 0-mercaptoethanol, reactivation was obtained as described above.Metal analyses were performed by atomic absorption spectrophotometry on a Perkin-Elmer 303 instrument. Before analy...

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Allosteric Interactions in Aspartate Transcarbamylase. II. Evidence for Different Conformational States of the Protein in the Presence and Absence of Specific Ligands

Abstract: In the previous paper it was shown that the

Cited by 324 publications

References 14 publications

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

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

Three-dimensional structures of aspartate carbamoyltransferase from Escherichia coli and of its complex with cytidine triphosphate.

Localization of the Zinc Binding Site of Aspartate Transcarbamoylase in the Regulatory Subunit

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