X-ray Scattering Titration of the Quaternary Structure Transition of Aspartate Transcarbamylase with a Bisubstrate Analogue: Influence of Nucleotide Effectors

Fetler, Luc; Tauc, Patrick; Hervé, Guy; Moody, Michael F.; Vachette, Patrice

doi:10.1006/jmbi.1995.0432

Cited by 49 publications

(68 citation statements)

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“…Are there conditions under which the T conformation would be predominant, or is this low-activity conformation inaccessible to the D236A ATCase? From previous work with the wild-type enzyme, it is known that addition of CTP, an allosteric inhibitor of the enzyme, shifts the T to R equilibrium by approximately 6-8% toward the T structure from a roughly equal concentration of the T and R states obtained in the presence of a subsaturating concentration of PALA (23). We therefore studied the effect of CTP on the unliganded mutant enzyme.…”

Section: Resultsmentioning

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: Resultsmentioning

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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“…The calculated curve is in excellent agreement with the experimental points with only a minor deviation observed for the largest values of [PALAIT,,. Though large, the ranges of acceptable values for each parameter clearly indicate the effects of the amino acid substitutions when compared with the values derived for the wild-type enzyme, namely K R = 20 nM for the catalytic subunit at pH 7 (Collins & Stark, 1971), L = 140, and c = 0.07 (Fetler et al, 1995). The behavior of the mutant enzymes in the presence of nucleotide effectors was investigated by adding ATP, CTP, or CTP plus UTP to a solution of either mutant already containing substrate analogues.…”

Section: Influence Of Allosteric Effectors On Wild-type and Mutant Homentioning

confidence: 88%

“…Samples of the wild-type and Glu-50/Ser-171 + Ala enzymes were prepared from stock solutions in 50 mM Tris-acetate buffer, pH 8.3 and 0.1 mM dithiothreitol as reported previously (Fetler et al, 1995). X-ray scattering curves were recorded on the smallangle scattering instrument D24 using synchrotron radiation at LURE-DCI, Orsay.…”

Section: Solution X-ray Scatteringmentioning

confidence: 99%

“…X-ray scattering curves were recorded on the smallangle scattering instrument D24 using synchrotron radiation at LURE-DCI, Orsay. The instrument (Depautex et al, 1987), the data acquisition system (Bordas et al, 1980), and the experimental procedures (Fetler et al, 1995) have been described.…”

Section: Solution X-ray Scatteringmentioning

confidence: 99%

“…The PALA titration data obtained with the Glu-SO/Ser-17 1 "+ Ala enzyme were analyzed in terms of the MWC model in a manner similar to that used for the wild-type enzyme by Fetler et al (1995), with the following differences. In the case of the wild-type enzyme, virtually all PALA molecules are bound at substoichiometric concentrations of PALA, because the active site concentration used (1 mM) is very large compared with the dissociation constant of PALA (about 20 nM for the catalytic subunit at pH 7 [Collins & Stark, 19711).…”

Section: Analysis Of Pal4 Titration Data For the Double Mutantmentioning

confidence: 99%

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The allosteric activator ATP induces a substrate‐dependent alteration of the quaternary structure of a mutant aspartate transcarbamoylase impaired in active site closure

1996

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Aspartate transcarbamoylase from Escherichia coli shows homotropic cooperativity for aspartate as well as heterotropic regulation by nucleotides. Structurally, it consists of two trimeric catalytic subunits and three dimeric regulatory subunits, each chain being comprised of two domains. Glu-50 and Ser-171 are involved in stabilizing the closed conformation of the catalytic chain. Replacement of Glu-50 or Ser-I71 by Ala in the holoenzyme has been shown previously to result in marked decreases in the maximal observed specific activity, homotropic cooperativity, and affinity for aspartate (Dembowski NJ, Newton CJ, Kantrowitz ER, 1990, Biochemistry 2937163723; Newton CJ, Kantrowitz ER, 1990, Biochemistry 29144k1451). We have constructed a double mutant enzyme combining both mutations. The resulting Glu-50/Ser-171 + Ala enzyme is 9-fold less active than the Ser-171 + Ala enzyme, 69-fold less active than the Glu-50 + Ala enzyme, and shows 1.3-fold and 1.6-fold increases in the [ S ] t 7 as compared to the Ser-171 + Ala and Glu-50 + Ala enzymes, respectively. However, the double mutant enzyme exhibits some enhancement of homotropic cooperativity with respect to aspartate, relative to the single mutant enzymes. At subsaturating concentrations of aspartate, the Glu-5O/Ser-171 -+ Ala enzyme is activated less by ATP than either the Glu-50 -+ Ala or Ser-171 -+ Ala enzyme, whereas CTP inhibition is intermediate between that of the two single mutants. As opposed to the wild-type enzyme, the Glu-SO/Ser-171 + Ala enzyme is activated by ATP and inhibited by CTP at saturating concentrations of aspartate. Structural analysis of the Ser-171 + Ala and Glu-50/Ser-171 + Ala enzymes by solution X-ray scattering indicates that both mutants exist in the same T quaternary structure as the wild-type enzyme in the absence of ligands, and in the same R quaternary structure in the presence of saturating N-(phosphonoacety1)-L-aspartate. However, saturating concentrations of carbamoyl phosphate and succinate are unable to convert a significant fraction of either mutant enzyme population to the R quaternary structure, as has been observed previously for the Glu-50 + Ala enzyme. The curves for both the Ser-171 -+ Ala and Glu-5O/Ser-171 -+ Ala enzymes obtained in the presence of substoichiometric amounts of PALA are linear combinations of the two extreme T and R states. The structural consequences of nucleotide binding to these two enzymes were also investigated. Most surprisingly, the direction and amplitude of the effect of ATP upon the double mutant enzyme were shown to vary depending upon the substrate analogue used.

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Large differences are observed between the crystal and solution quaternary structures of allosteric aspartate transcarbamylase in the R state

et al. 1997

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Solution scattering curves evaluated from the crystal structures of the T and R states of the allosteric enzyme aspartate transcarbamylase from Escherichia coli were compared with the experimental x-ray scattering patterns. Whereas the scattering from the crystal structure of the T state agrees with the experiment, large deviations reflecting a significant difference between the quaternary structures in the crystal and in solution are observed for the R state. The experimental curve of the R state was fitted by rigid body movements of the subunits in the crystal R structure which displace the latter further away from the T structure along the reaction coordinates of the T-->R transition observed in the crystals. Taking the crystal R structure as a-reference, it was found that in solution the distance between the catalytic trimers along the threefold axis is 0.34 nm larger and the trimers are rotated by 11 degrees in opposite directions around the same axis; each of the three regulatory dimers is rotated by 9 degrees around the corresponding twofold axis and displaced by 0.14 nm away from the molecular center along this axis.

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X-ray Scattering Titration of the Quaternary Structure Transition of Aspartate Transcarbamylase with a Bisubstrate Analogue: Influence of Nucleotide Effectors

Cited by 49 publications

References 0 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

The allosteric activator ATP induces a substrate‐dependent alteration of the quaternary structure of a mutant aspartate transcarbamoylase impaired in active site closure

Large differences are observed between the crystal and solution quaternary structures of allosteric aspartate transcarbamylase in the R state

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