Carbamyl Phosphate Modifies the <i>T</i> Quaternary Structure of Aspartate Transcarbamylase, thereby Facilitating the Structural Transition Associated with Cooperativity

Tauc, Patrick; Vachette, Patrice

doi:10.1107/s0021889897001866

Cited by 15 publications

(25 citation statements)

References 18 publications

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“…Previous sedimentation velocity measurements were interpreted by assuming this equilibrium, although the very careful analysis of boundary spreading in this kind of experiment by the same authors were clearly in favor of a two-state transition (25). Similarly, our previous SAXS titration studies of the transition using PALA, alone or in the presence of the first substrate or nucleotide effectors, as well as time-resolved SAXS studies of the transition, also led us to conclude firmly to the existence of a concerted transition between T and R conformations (23,(26)(27)(28). However, to date, there had been no direct proof of the existence of the structure equilibrium for an unliganded enzyme, i.e., of the accessibility of an R-like structure to an unliganded enzyme.…”

Section: Direct Evidence Of a Thermodynamic Equilibrium Between Diffementioning

confidence: 99%

Direct observation in solution of a preexisting structural equilibrium for a mutant of the allosteric aspartate transcarbamoylase

Fetler

Kantrowitz²,

Vachette³

2007

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

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Many signaling and metabolic pathways rely on the ability of some of the proteins involved to undergo a substrate-induced transition between at least two structural states. Among the various models put forward to account for binding and activity curves of those allosteric proteins, the Monod, Wyman, and Changeux model for allostery theory has certainly been the most influential, although a central postulate, the preexisting equilibrium between the lowactivity, low-affinity quaternary structure and the high-activity, high-affinity quaternary structure states in the absence of substrates, has long awaited direct experimental substantiation. Upon substrate binding, allosteric Escherichia coli aspartate transcarbamoylase adopts alternate quaternary structures, stabilized by a set of interdomain and intersubunit interactions, which are readily differentiated by their solution x-ray scattering curves. Disruption of a salt link, which is observed only in the low-activity, lowaffinity quaternary structure, between Lys-143 of the regulatory chain and Asp-236 of the catalytic chain yields a mutant enzyme that is in a reversible equilibrium between at least two states in the absence of ligand, a major tenet of the Monod, Wyman, and Changeux model. By using this mutant as a magnifying glass of the structural effect of ligand binding, a comparative analysis of the binding of carbamoyl phosphate (CP) and analogs points out the crucial role of the amine group of CP in facilitating the transition toward the high-activity, high-affinity quaternary state. Thus, the cooperative binding of aspartate in aspartate transcarbamoylase appears to result from the combination of the preexisting quaternary structure equilibrium with local changes induced by CP binding.enzyme regulation ͉ homotropic cooperativity ͉ induced fit ͉ intersubunit interactions ͉ small-angle x-ray scattering M any cellular responses, such as cell signaling, cell movements, intermediary metabolism, and the selective expression of genes, rely on the capacity of proteins to switch between different stable conformations in a reversible manner (1). Crystallographic studies have shown that, in agreement with the Monod, Wyman, and Changeux model for allostery theory (MWC) (2), most such proteins are made up of a finite number of identical subunits regularly organized around symmetry axes, which increases the stability of their conformational states and enhances the abrupt, switch-like character of their quaternarystructure transition between a low-activity, low-affinity (T) state and a high-activity, high-affinity (R) state (3). A critical statement of the MWC was that this conformational transition involves states that are populated in the absence of ligand and may spontaneously interconvert, the ligand stabilizing the conformation to which it binds with higher affinity. Initially, these concepts were developed based on catalytic activity measurements performed with regulatory enzymes, such as aspartate transcarbamoylase from Escherichia coli (ATCase). Most of these data...

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Section: Direct Evidence Of a Thermodynamic Equilibrium Between Diffementioning

confidence: 99%

Direct observation in solution of a preexisting structural equilibrium for a mutant of the allosteric aspartate transcarbamoylase

Fetler

Kantrowitz²,

Vachette³

2007

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

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“…Small-angle x-ray scattering (12) and sedimentation velocity (13) studies have shown that CP does not cause significant global quaternary changes, whereas local changes are observed by circular dichroism in the aromatic region (14). These data suggest that upon CP binding, local conformational changes occur that create a viable Asp-binding site.…”

mentioning

confidence: 99%

Structural basis for ordered substrate binding and cooperativity in aspartate transcarbamoylase

Wang

Stieglitz²,

Cardia³

et al. 2005

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

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X-ray structures of aspartate transcarbamoylase in the absence and presence of the first substrate carbamoyl phosphate are reported. These two structures in conjunction with in silico docking experiments provide snapshots of critical events in the function of the enzyme. The ordered substrate binding, observed experimentally, can now be structurally explained by a conformational change induced upon the binding of carbamoyl phosphate. This induced fit dramatically alters the electrostatics of the active site, creating a binding pocket for aspartate. Upon aspartate binding, a further change in electrostatics causes a second induced fit, the domain closure. This domain closure acts as a clamp that both facilitates catalysis by approximation and also initiates the global conformational change that manifests homotropic cooperativity.allosteric transition ͉ induced fit ͉ homotropic cooperativity E nzymes that exhibit positive cooperativity play a vital role in the regulation of the rates of key metabolic pathways by amplifying the response of a pathway to an effector molecule. In the case of aspartate transcarbamoylase (ATCase), substrateinduced domain closure triggers a quaternary conformational change that results in the observed homotropic cooperativity, one mechanism by which this enzyme controls the rate of de novo pyrimidine biosynthesis. Understanding the molecular features of domain closure not only provides insights into the mechanism of homotropic cooperativity but also demonstrates how ligandinduced domain closure can be used as part of catalytic mechanism of many enzymes (1, 2).The Escherichia coli ATCase is composed of six chains (M r 34,000 each) grouped into two trimeric catalytic subunits and six chains (M r 17,000 each) grouped into three dimeric regulatory subunits. The three active sites in the catalytic subunit are shared across the interface between adjacent chains (3, 4), whereas the regulatory subunits contain the binding sites for the heterotropic activator, ATP, as well as the heterotropic inhibitors, CTP and UTP. Each catalytic chain is composed of two structural domains, the carbamoyl phosphate (CP) domain (residues 1-135 and 292-310) and the L-aspartate (Asp) domain (residues 136-291), which contain the binding sites for CP and Asp, respectively. Each regulatory chain is also composed of two structural domains, the AL domain (residues 1-100) and the Zn domain (residues 101-153), which contain the binding sites for the allosteric effectors and the structural Zn, respectively. In mammals ATCase exists as a component of the multienzyme complex CAD (carbamoyl phosphate synthetase, aspartate transcarbamoylase, and dihydroorotase) (5), and in humans, CAD has become a target for the development of antiproliferation drugs (6). The E. coli enzyme and the ATCase portion of CAD are 44% conserved, and all residues known to be involved in substrate binding and catalysis are conserved. Domain closure is triggered by the ordered binding of the substrates (7), with CP binding before Asp, and N-carbamoyl-L-...

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“…the variation of the fraction of molecules in the R-state versus the active site occupancy. Similar titration experiments were performed using succinate, a competitive inhibitor of aspartate, in the presence of saturating amounts of carbamoyl phosphate, the first physiological substrate [49]. Also in this case only two conformers were detected as in the classical concerted allosteric model proposed by Monod et al, [50].…”

Section: Study Of Allosteric Transitionsmentioning

confidence: 70%

“…The radius of gyration increases by about 5% upon the T to R transition. Using the SAXS approach it has been possible to perform titration experiments [48,49] whereby a series of patterns were recorded at a variable concentration of substrate. Using PALA (the bisubstrate analogue already mentioned) it has been possible to follow the transition from T to R state and to establish that it only involves the two extreme structures T and R, and no intermediate species can be detected.…”

Section: Study Of Allosteric Transitionsmentioning

confidence: 99%

Small Angle X-Ray Scattering: A Powerful Tool to Analyze Protein Conformation in Solution

Dainese¹,

Sabatucci²,

Cozzani³

2005

COC

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In the field of molecular biology and biochemistry in which structural genomics comes as a complement to genome sequencing, Small-Angle X-ray Scattering (SAXS) is an experimental technique that, though not widely known and applied, represents a very powerful tool in the framework of post-genome structural studies. The aim of this review is to present a synthetic description of the basic principles of the theory of SAXS and to discuss in more detail its applications to the study of different biological molecules, focusing the attention to the recent advances in the structural analysis of proteins. These studies are presently undergoing a spectacular expansion associated with the development of powerful data analysis software, with the improvement of the quality of data recorded with synchrotron radiation, and finally with the increasing availability of high resolution three-dimensional structures which can constitute a starting point for the analysis of protein conformations in solution. The advantage of SAXS with respect to other techniques in the structural studies of proteins resides in the possibility of performing the measurements in any desired solvent and in the ability to follow changes of the protein structure which may occur as a response to a variety of stimuli: pH or temperature changes, interaction with small ligands, influence of substrate analogues, chemical or genetic modifications, etc. In this review we describe the principles of SAXS and the most recent methods for data analysis in the field of structural biology and, finally, we report some examples of the use of this technique as a powerful tool to the structural study of proteins in solution.

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Carbamyl Phosphate Modifies the T Quaternary Structure of Aspartate Transcarbamylase, thereby Facilitating the Structural Transition Associated with Cooperativity

Cited by 15 publications

References 18 publications

Direct observation in solution of a preexisting structural equilibrium for a mutant of the allosteric aspartate transcarbamoylase

Direct observation in solution of a preexisting structural equilibrium for a mutant of the allosteric aspartate transcarbamoylase

Structural basis for ordered substrate binding and cooperativity in aspartate transcarbamoylase

Small Angle X-Ray Scattering: A Powerful Tool to Analyze Protein Conformation in Solution

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