Structure and arrangement of the regulatory subunits in aspartate transcarbamylase

Cohlberg, Jeffrey A.; Pigiet, Vincent; Schachman, H. K.

doi:10.1021/bi00768a013

Cited by 124 publications

(76 citation statements)

References 34 publications

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“…The quaternary structure of this enzyme results from the association of two catalytic c3 subunits (trimers of catalytic chains of 310 amino acids; molecular weight, 34,301) held together through their interactions with three regulatory r2 subunits (dimers of regulatory chains of 152 amino acids; molecular weight, 17,109) (8). Both types of chains are organized in two domains, the carbamylphosphate and the aspartate binding domains in the case of the catalytic chain and the allosteric and the zinc (Zn) domains in the case of the regulatory chain.…”

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Aspartate transcarbamylase from the deep-sea hyperthermophilic archaeon Pyrococcus abyssi: genetic organization, structure, and expression in Escherichia coli

et al. 1997

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The genes coding for aspartate transcarbamylase (ATCase) in the deep-sea hyperthermophilic archaeon Pyrococcus abyssi were cloned by complementation of a pyrB Escherichia coli mutant. The sequence revealed the existence of a pyrBI operon, coding for a catalytic chain and a regulatory chain, as in Enterobacteriaceae.Comparison of primary sequences of the polypeptides encoded by the pyrB and pyrI genes with those of homologous eubacterial and eukaryotic chains showed a high degree of conservation of the residues which in E. coli ATCase are involved in catalysis and allosteric regulation. The regulatory chain shows more-extensive divergence with respect to that of E. coli and other Enterobacteriaceae than the catalytic chain. Several substitutions suggest the existence in P. abyssi ATCase of additional hydrophobic interactions and ionic bonds which are probably involved in protein stabilization at high temperatures. The catalytic chain presents a secondary structure similar to that of the E. coli enzyme. Modeling of the tridimensional structure of this chain provides a folding close to that of the E. coli protein in spite of several significant differences. Conservation of numerous pairs of residues involved in the interfaces between different chains or subunits in E. coli ATCase suggests that the P. abyssi enzyme has a quaternary structure similar to that of the E. coli enzyme. P. abyssi ATCase expressed in transgenic E. coli cells exhibited reduced cooperativity for aspartate binding and sensitivity to allosteric effectors, as well as a decreased thermostability and barostability, suggesting that in P. abyssi cells this enzyme is further stabilized through its association with other cellular components.Studies of extremophilic organisms provide information on the molecular mechanisms of biological adaptation to extreme environments. In this regard, Pyrococcus abyssi is of particular interest, being a hyperthermophilic and barophilic/barotolerant archaeon. This euryarchaebacterium, isolated from a deepsea hydrothermal vent located 2,000 m deep in the North Fiji Basin, was characterized by Erauso et al. (15,16). It is a heterotrophic and strictly anaerobic microorganism and belongs to the sulfur-metabolizing group. At atmospheric pressure, it grows at 67 to 102°C, with an optimum at 96°C. Hydrostatic pressure slightly increases the growth rate of P. abyssi as well as its optimal and maximal growth temperatures (16).Aspartate transcarbamylase (ATCase) (EC 2.1.3.2) is the first enzyme of the pyrimidine biosynthetic pathway. It catalyzes the carbamylation of the amino group of aspartate by carbamylphosphate with formation of carbamylaspartate and phosphate. ATCase is extensively studied as a model for structure-function relationships in cooperativity and allosteric regulation mechanisms, as well as for the evolution of these mechanisms from prokaryotes to humans. The structure and properties of the Escherichia coli enzyme have been studied in detail (for reviews, see references 3, 10, 26, 32, 40, and 70).This dodecame...

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Aspartate transcarbamylase from the deep-sea hyperthermophilic archaeon Pyrococcus abyssi: genetic organization, structure, and expression in Escherichia coli

et al. 1997

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“…It is now known that it is composed of six catalytic (c) and six regulatory (r) polypeptide chains (4)(5)(6)(7) arranged in a molecule having 2-fold and 3-fold axes of symmetry (8)(9)(10)(11). The catalytic chains are organized as trimers (C) both in the intact enzyme and after its dissociation with certain mercurials (5).…”

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Aspartate Transcarbamoylase Molecules Lacking One Regulatory Subunit

Yang

Syvänen

Nagel

et al. 1974

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

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Reconstitution of aspartate transcarbamoylase (EC 2.1.3.2) from dilute solutions of the isolated regulatory and catalytic subunits, with the latter in large excess, led to the formation of appreciable amounts of a second, stable component in addition to the reconstituted enzyme. The purified component, designated r4c6, was found to have a molecular weight about 3 X 104. less than that of the native enzyme, and it combined with isolated regulhtory subunit to form aspartate transcarbamoylase.It also combined with one succinylated regulatory subunit to form a hybrid species that was identified electrophoretically. These findings indicate that r4cs differs from the native enzyme in that only two (rather than three) regulatory subunits participate in "crosslinking" the two catalytic trimers. The "incomplete" enzyme, r4c6, exhibits the characteristic sigmoidal saturation behavior and CTP inhibition of aspartate transcarbamoylase; however these allosteric effects are reduced in extent by about one-third in comparison to the native enzyme and free catalytic subunits. The complex, which may be an intermediate in the assembly and dissociation of the native enzyme, is useful in assessing the role of the various bonding domains responsible for the stability and regulatory properties of the native enzyme.Following the discovery that the regulatory enzyme, aspartate transcarbamoylase (EC 2.1.3.2; carbamoylphosphate: 'A aspartate carbamoyltransferase) from Escherichia coli, is composed of discrete subunits for catalysis and regulation, each containing a unique polypeptide chain (1), there have been many studies aimed at determining the structure and mechanism of action of the enzyme (2, 3). It is now known that it is composed of six catalytic (c) and six regulatory (r) polypeptide chains (4-7) arranged in a molecule having 2-fold and 3-fold axes of symmetry (8-11). The catalytic chains are organized as trimers (C) both in the intact enzyme and after its dissociation with certain mercurials (5). Although the C subunits show little tendency to associate to form discrete species, they combine readily with free regulatory subunits (R), yielding reconstituted molecules with the unusual physical and kinetic properties of the native enzyme (1, 12). The R subunits, as a result, have been viewed as "crosslinks" that bind the two C subunits in the intact enzyme molecules. Various experiments have shown that the r chains are organized as dimers both in the native enzyme and in the R subunit released by treating the enzyme with mercurials (7). In addition, considerations of the weight composition of the enzyme in terms of c and r chains (1, 6, 7) plus hybridization experiments with native C and mixtures of native and succinylated R (13) have led to the conclusion that there are three R dimers in each enzyme molecule. Recent evidence from electron microscopy (14) and x-ray diffraction studies (10, 11) has provided support for a model of the enzyme as a complex containing two catalytic trimers bonded through three regulatory dimers;...

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“…In a similar experiment with *ATCase in the low ionic strength buffer containing PALA and 0.I mM Zn2+ to stabilize the R dimers (17), there was a significant but slow exchange of R subunits. After incubation of *ATCase with free R subunits for 0, 5, 20, 60, 90, and 120 min, the amounts of radioactivity associated with the free R subunits were 1.6, 2.2, 3.4, 4.4, 4.7, and 5.2%, respectively.…”

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“…The native enzyme is composed of two catalytic trimers (C) and three regulatory dimers (R) with each catalytic polypeptide chain (c) in one C subunit linked to a c chain in the other C subunit via an R subunit (13)(14)(15)(16)(17). In this structure, designated C2R3 or c6r6, there are six c-c bonding domains within the two C trimers, three r-r domains linking the regulatory polypeptide chains, r, within the three R dimers, and six c-r domains connecting the c and r chains (18).…”

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“…Intact ATCase labeled randomly in both the C and R subunits (*ATCase) with Exchange reactions were performed at 250 with *R at 2.6 MM, ATCase at 0.17 AM, and PALA at 55 MM. The low and high ionic strength buffers were the same as those described in Table 1 except that 0.1 mM Zn2+ was also present in order to stabilize the R dimers (17). The experimental procedures were the same as those for Table 1.…”

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Ligand-promoted weakening of intersubunit bonding domains in aspartate transcarbamoylase

Subramani

Bothwell

Gibbons

et al. 1977

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

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The cooperativity and feedback inhibition exhibited by the It is generally recognized that the homotropic and heterotropic effects exhibited by allosteric enzymes are attributable to ligand-promoted conformational changes whereby the proteins are converted from a constrained or low-affinity state to a relaxed form having a higher affinity for substrates (1, 2). Since these conformational changes are mediated by alterations in the subunit interactions, it is important to determine the strengths of the intersubunit bonding domains and their perturbation by ligands (3). Despite the evident need for such data, quantitative estimates of ligand effects are available for only a few proteins. Of these, hemoglobin has been studied most extensively. The dissociation constants for the tetramer-dimer equilibria have been evaluated for hemoglobin in both the oxy and deoxy states (4-9), and the changes in the intersubunit contact energies have been related to the functional properties of the protein (10, 11). In this paper we present an approach aimed at providing comparable information for the regulatory enzyme, aspartate transcarbamoylase (ATCase; carbamoyl- (20).Since the dissociation of ATCase into free subunits could not be measured directly, we initiated studies of subunit exchange as an alternative, sensitive approach for estimating the strengths of the intersubunit bonding domains. Very little exchange of either C or R subunits occurred even in the presence of the bisubstrate analog, N-(phosphonacetyl)-L-aspartate (PALA), which promotes the conformational transition of ATCase to the relaxed state (20,21). Studies were conducted, therefore, on the effect of this active-site ligand on the bonding domains in ATCase-like molecules lacking one R subunit (22)(23)(24)(25). This R-deficient species, designated C2R2, which exhibits both cooperativity and inhibition by CTP (22,23), is an intermediate in both the in vitro assembly of ATCase from subunits and the dissociation of the enzyme (24,26

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Structure and arrangement of the regulatory subunits in aspartate transcarbamylase

Cited by 124 publications

References 34 publications

Aspartate transcarbamylase from the deep-sea hyperthermophilic archaeon Pyrococcus abyssi: genetic organization, structure, and expression in Escherichia coli

Aspartate transcarbamylase from the deep-sea hyperthermophilic archaeon Pyrococcus abyssi: genetic organization, structure, and expression in Escherichia coli

Aspartate Transcarbamoylase Molecules Lacking One Regulatory Subunit

Ligand-promoted weakening of intersubunit bonding domains in aspartate transcarbamoylase

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