Glu‐50 in the catalytic chain of <i>Escherichia coli</i> aspartate transcarbamoylase plays a crucial role in the stability of the R quaternary structure

Tauc, Patrick; Keiser, R. T.; Kantrowitz, Evan R.; Vachette, Patrice

doi:10.1002/pro.5560031112

Cited by 16 publications

(21 citation statements)

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“…Although the unligated E50A holoenzyme and the PALA ligated E50A holoenzyme exhibit the same T and R state structures, respectively, as the wild-type enzyme, the combination of the natural substrate carbamoyl phosphate and the aspartate analog succinate does not induce a structural change (12). This is quite different from what is observed for the wild-type enzyme in which case this combination of substrate and substrate analog induces the complete structural transition to the R state (12).…”

Section: Aspartate Saturation Curves Of the (At-c) 2 R 3 (E50a-c) 2mentioning

confidence: 73%

“…Small Angle X-ray Scattering-The importance of the interdomain bridging interactions involving E50A for the function of aspartate transcarbamoylase has been established by small angle x-ray scattering (12). Although the unligated E50A holoenzyme and the PALA ligated E50A holoenzyme exhibit the same T and R state structures, respectively, as the wild-type enzyme, the combination of the natural substrate carbamoyl phosphate and the aspartate analog succinate does not induce a structural change (12).…”

Section: Aspartate Saturation Curves Of the (At-c) 2 R 3 (E50a-c) 2mentioning

confidence: 99%

“…The unliganded E50A enzyme and the PALA-ligated E50A exist in the same T and R state conformations, respectively, as does the wild-type enzyme (12). However, carbamoyl phosphate plus succinate induces only a small change in the T state structure, reflecting only binding and no quaternary conformational change.…”

mentioning

confidence: 92%

“…To investigate the structural effect of breaking the Glu 50 interactions, SAXS (12) and time-resolved small angle scattering (TR-SAXS) (13) were employed. The unliganded E50A enzyme and the PALA-ligated E50A exist in the same T and R state conformations, respectively, as does the wild-type enzyme (12).…”

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confidence: 99%

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Domain Bridging Interactions

Sakash¹,

Williams²,

Tsuruta³

et al. 2001

Journal of Biological Chemistry

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Aspartate transcarbamoylase undergoes a domain closure in the catalytic chains upon binding of the substrates that initiates the allosteric transition. Interdomain bridging interactions between Glu 50 and both Arg 167 and Arg 234 have been shown to be critical for stabilization of the R state. A hybrid version of the enzyme has been generated in vitro containing one wildtype catalytic subunit, one catalytic subunit in which Glu 50 in each catalytic chain has been replaced by Ala (E50A), and wild-type regulatory subunits. Thus, the hybrid enzyme has one catalytic subunit capable of domain closure and one catalytic subunit incapable of domain closure. The hybrid does not behave as a simple mixture of the constituent subunits; it exhibits lower catalytic activity and higher aspartate affinity than would be expected. As opposed to the wild-type enzyme, the hybrid is inhibited allosterically by CTP at saturating substrate concentrations. As opposed to the E50A holoenzyme, the hybrid is not allosterically activated by ATP at saturating substrate concentrations. Small angle x-ray scattering showed that three of the six interdomain bridging interactions in the hybrid is sufficient to cause the global structural change to the R state, establishing the critical nature of these interactions for the allosteric transition of aspartate transcarbamoylase.Escherichia coli aspartate transcarbamoylase catalyzes the committed step of pyrimidine biosynthesis, the condensation of carbamoyl phosphate and L-aspartate to form N-carbamoyl-Laspartate (1). The enzyme shows homotropic cooperativity for the substrate L-aspartate and is heterotropically regulated by ATP, CTP (1), and UTP in the presence of CTP (2). The holoenzyme is composed of six catalytic chains (M r 34,000), associated as two trimeric subunits, and six regulatory chains (M r 17,000), associated as three dimeric subunits. The active sites, three/ trimer, are located at the interface between catalytic chains, whereas the allosteric sites, two/dimer, are located on the Nterminal domain of the regulatory chains.Two functionally and structurally different states of aspartate transcarbamoylase have been characterized. The low affinity, low activity conformation of the enzyme is described as the "tense" or T state, whereas the high affinity, high activity conformation of the enzyme is described as the "relaxed" or R state. The conversion from the T state to the R state occurs upon aspartate binding to the enzyme in the presence of carbamoyl phosphate. Structurally, the enzyme elongates by at least 11 Å along the 3-fold axis, and the catalytic trimers and regulatory dimers rotate along their axes of symmetry, 10°and 15°, respectively (3). The allosteric transition can also be monitored in solution using small angle x-ray scattering (SAXS) 1 (4). In addition to the quaternary changes, several tertiary changes also occur because of a rearrangement of several important inter-and intrasubunit interactions. One important intrasubunit interaction that stabilizes the R state is between...

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Section: Aspartate Saturation Curves Of the (At-c) 2 R 3 (E50a-c) 2mentioning

confidence: 73%

Section: Aspartate Saturation Curves Of the (At-c) 2 R 3 (E50a-c) 2mentioning

confidence: 99%

mentioning

confidence: 92%

mentioning

confidence: 99%

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Domain Bridging Interactions

Sakash¹,

Williams²,

Tsuruta³

et al. 2001

Journal of Biological Chemistry

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“…Because the interaction between Asp-100 and Arg-65 is maintained in both the T and R structures (Ke et al, 1988;Stevens et al, 1990), and because neither residue has any direct role in substrate binding or catalysis, it was suspected that the increase in activity, affinity for aspartate, and cooperativity observed for the Asp-100 -+ Ala' enzyme might be due to promotion of domain closure induced by the weakening of the C1LC2 interface. To test this hypothesis, we constructed a double mutant of aspartate transcarbamoylase in which the Asp-100 --t Ala mutation was introduced into the Glu-50 + Ala holoenzyme, a mutant in which domain closure is impaired (Newton & Kantrowitz, 1990;Tauc et al, 1994).Here we report the kinetic and structural characterization of the Glu-50/Asp-100 + Ala5 double mutant of aspartate transcarbamoylase and its comparison with the wild-type, Glu-50 -+ Ala, and Asp-100 -+ Ala holoenzymes. …”

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confidence: 99%

Weakening of the interface between adjacent catalytic chains promotes domain closure in escherichia coli aspartate transcarbamoylase

et al. 1995

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Aspartate transcarbamoylase from Escherichia coli is a dodecameric enzyme consisting of two trimeric catalytic subunits and three dimeric regulatory subunits. Asp-100, from one catalytic chain, is involved in stabilizing the C1-C2 interface by means of its interaction with Arg-65 from an adjacent catalytic chain. Replacement of Asp-100 by Ala has been shown previously to result in increases in the maximal specific activity, homotropic cooperativity, and the affinity for aspartate (Baker DP, Kantrowitz ER, 1993, Biochemistry 32:10150-10158). In order to determine whether these properties were due to promotion of domain closure induced by the weakening of the C1-C2 interface, we constructed a double mutant version of aspartate transcarbamoylase in which the Asp-100 + Ala mutation was introduced into the Glu-50 -+ Ala holoenzyme, a mutant in which domain closure is impaired.The Glu-5O/Asp-100 + Ala enzyme is fourfold more active than the Glu-50 -+ Ala enzyme, and exhibits significant restoration of homotropic cooperativity with respect to aspartate. In addition, the Asp-IO0 -+ Ala mutation restores the ability of the Glu-50 -+ Ala enzyme to be activated by succinate and increases the affinity of the enzyme for the bisubstrate analogue N-(phosphonacety1)-L-aspartate (PALA). At subsaturating concentrations of aspartate, the Glu-50/Asp-100 -+ Ala enzyme is activated more by ATP than the Glu-50 + Ala enzyme and is also inhibited more by CTP than either the wild-type or the Glu-50 + Ala enzyme. As opposed to the wild-type enzyme, the Glu-50/Asp-100 + Ala enzyme is activated by ATP and inhibited by CTP at saturating concentrations of aspartate. Structural analysis of the Glu-50/Asp-100 + Ala enzyme by solution X-ray scattering indicates that the double mutant exists 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 PALA. However, saturating concentrations of carbamoyl phosphate and succinate only convert a fraction of the Glu-50/Asp-100 -+ Ala enzyme population to the R quaternary structure, a behavior intermediate between that observed for the Glu-50 + Ala and wild-type enzymes. Solution X-ray scattering was also used to investigate the structural consequences of nucleotide binding to the Glu-50/Asp-100 + Ala enzyme.Keywords: domain closure; homotropic cooperativity; protein structure-function; site-specific mutagenesis; solution X-ray scattering *We dedicate this paper to the memory of our friend and colleague Escherichia coli aspartate transcarbamoylase (ATCase; EC Frederick C. Wedler, whose work on the catalytic mechanism of aspar-2.1.3.2) catalyzes the committed step in the biosynthesis of pytate transcarbamoylase has been fundamental to the understanding of rimidine nucleotides: the reaction between carbamoyl phosphate this complex enzyme. His personal and intellectual contributions to the scientific community will be sorely missed.and L-aspartate to form N-carbamoyl-L-aspartate and inorganic Reprint requ...

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Direct observation of an altered quaternary-structure transition in a mutant aspartate transcarbamoylase

1998

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Time-resolved small-angle X-ray scattering (TR-SAXS) was used to monitor the structural changes that occur upon the binding of the natural substrates to a mutant version of the allosteric enzyme aspartate transcarbamoylase from Escherichia coli, in which the creation of a critical link stabilizing the R state of the enzyme is hindered. Previously, SAXS experiments at equilibrium showed that the structures of the unligated mutant enzyme and the mutant enzyme saturated with a bisubstrate analog are indistinguishable from the T and R state structures, respectively, of the wild-type enzyme (Tauc et al., Protein Sci. 3:1998-2004, 1994). However, as opposed to the wild-type enzyme, the combination of one substrate, carbamoyl phosphate, and succinate, an analog of aspartate, did not convert the mutant enzyme into the R state. By using TR-SAXS we have been able to study the transient steady-state during catalysis using the natural substrates rather than the nonreactive substrate analogs. The steady-state in the presence of saturating amount of substrates is a mixture of 60% T and 40% R structures, which is further converted entirely to R in the additional presence of ATP. These results provide a structural explanation for the reduced cooperativity observed with the mutant enzyme as well as for the stimulation by ATP at saturating concentrations of substrates. They also illustrate the crucial role played by domain motions and quaternary-structure changes for both the homotropic and heterotropic aspects of allostery.

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Glu‐50 in the catalytic chain of Escherichia coli aspartate transcarbamoylase plays a crucial role in the stability of the R quaternary structure

Cited by 16 publications

References 28 publications

Domain Bridging Interactions

Domain Bridging Interactions

Weakening of the interface between adjacent catalytic chains promotes domain closure in escherichia coli aspartate transcarbamoylase

Direct observation of an altered quaternary-structure transition in a mutant aspartate transcarbamoylase

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