The conserved residues glutamate-37, aspartate-100, and arginine-269, are important for the structural stabilization of Escherichia coli aspartate transcarbamoylase

Baker, Darren P.; Kantrowitz, Evan R.

doi:10.1021/bi00089a034

Cited by 32 publications

(27 citation statements)

References 59 publications

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“…The uracil-containing single-stranded DNA required was obtained by infection of E. coli strain CJ236, containing the phagemid pEK54 (17) for the C47A mutation and the phagemid pEK152 (18) for the A241C mutation, with the helper phage M13K07 (19). Selection of the mutations was performed by sequencing with single-stranded DNA isolated from phagemid candidates after coinfection with the helper phage M13K07 (19).…”

Section: Chemicalsmentioning

confidence: 99%

Stabilization of the R Allosteric Structure of Escherichia coli Aspartate Transcarbamoylase by Disulfide Bond Formation

West

Tsuruta

Kantrowitz

2002

Journal of Biological Chemistry

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Here we report the first use of disulfide bond formation to stabilize the R allosteric structure of Escherichia coli aspartate transcarbamoylase. In the R allosteric state, residues in the 240s loop from two catalytic chains of different subunits are close together, whereas in the T allosteric state they are far apart. By substitution of Ala-241 in the 240s loop of the catalytic chain with cysteine, a disulfide bond was formed between two catalytic chains of different subunits. The cross-linked enzyme did not exhibit cooperativity for aspartate. The maximal velocity was increased, and the concentration of aspartate required to obtain onehalf the maximal velocity, [Asp] 0.5 , was reduced substantially. Furthermore, the allosteric effectors ATP and CTP did not alter the activity of the cross-linked enzyme. When the disulfide bonds were reduced by the addition of 1,4-dithio-DL-threitol the resulting enzyme had kinetic parameters very similar to those observed for the wild-type enzyme and regained the ability to be activated by ATP and inhibited by CTP. Small-angle x-ray scattering was used to verify that the cross-linked enzyme was structurally locked in the R state and that this enzyme after reduction with 1,4-dithio-DL-threitol could undergo an allosteric transition similar to that of the wild-type enzyme. The complete abolition of homotropic and heterotropic regulation from stabilizing the 240s loop in its closed position in the R state, which forms the catalytically competent active site, demonstrates the significance that the quaternary structural change and closure of the 240s loop has in the functional mechanism of aspartate transcarbamoylase.Escherichia coli aspartate transcarbamoylase (EC 2.1.3.2) is one of the best characterized allosteric enzymes, so it serves an important role in the study of allosteric regulation. This enzyme catalyzes the committed step of pyrimidine biosynthesis, the carbamoylation of the amino group of L-aspartate by carbamoyl phosphate to form N-carbamoyl-L-aspartate (1). The enzyme demonstrates homotropic cooperativity for the substrate Laspartate and is heterotropically regulated by effectors ATP, CTP (1), and UTP in the presence of CTP (2). Aspartate transcarbamoylase from E. coli is a dodecamer composed of six catalytic chains organized as two trimeric subunits and six regulatory chains organized as three dimeric subunits. Because aspartate transcarbamoylase is so well characterized and serves as a model for allosteric enzymes it is of particular interest to thoroughly understand the molecular mechanism of allostery of this enzyme.Two different structural and functional states of aspartate transcarbamoylase have been characterized (3-6). The low activity, low affinity conformation of the enzyme is called the T state, and the high activity, high affinity conformation of the enzyme is called the R state. The conversion from the T to the R state occurs upon aspartate binding to the holoenzyme in the presence of carbamoyl phosphate, which is the source of the observed homotropic co...

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Section: Chemicalsmentioning

confidence: 99%

Stabilization of the R Allosteric Structure of Escherichia coli Aspartate Transcarbamoylase by Disulfide Bond Formation

West

Tsuruta

Kantrowitz

2002

Journal of Biological Chemistry

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“…2). Because succinate is an aspartate analogue, this apparent increase in the affinity for succinate presumably reflects the increase in affinity that the Asp-100 + Ala enzyme shows for aspartate (Baker & Kantrowitz, 1993). By contrast, the Glu-50 + Ala enzyme cannot be activated (Fig.…”

Section: Discussionmentioning

confidence: 97%

“…These interdomain bridging interactions have been shown to be important for the establishment of the high-affinity, high-activity conformation of the active site (Newton & Kantrowitz, 1990), as well as for the stabilization of the R quaternary structure , most likely through their role in the closure of the two domains of the catalytic chain. In contrast to the Glu-50 +Ala substitution, replacement of Asp-100 by Ala results in an enzyme that exhibits increased homotropic cooperativity, activity, and affinity for aspartate (Baker & Kantrowitz, 1993). Because the replacement of Asp-100 by Ala facilitates domain closure at lower aspartate concentrations (Table l), it was suspected that the addition of the Asp-100 + Ala mutation to the Glu-50 + Ala enzyme might increase homotropic cooperativity.…”

Section: Discussionmentioning

confidence: 99%

“…Recently, Baker and Kantrowitz (1993) showed that His-41, Asp-100, and Asp-90 from C1, which form interactions with Glu-37, Arg-65, and Arg-269 from C2, respectively, are important for the structural stabilization of the catalytic subunit. Interestingly, replacement of Asp-100 by Ala results in a mutant that exhibits a higher specific activity than the wild-type holoenzyme, as well as a twofold increase in the affinity for aspartate and an increase in homotropic cooperativity (Baker & Kantrowitz, 1993). 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.…”

Section: Introductionmentioning

confidence: 99%

“…Each chain within the catalytic subunit is tethered to its neighbor by a complex network of interactions that forms the CI-CZ4 interface. Recently, Baker and Kantrowitz (1993) showed that His-41, Asp-100, and Asp-90 from C1, which form interactions with Glu-37, Arg-65, and Arg-269 from C2, respectively, are important for the structural stabilization of the catalytic subunit. Interestingly, replacement of Asp-100 by Ala results in a mutant that exhibits a higher specific activity than the wild-type holoenzyme, as well as a twofold increase in the affinity for aspartate and an increase in homotropic cooperativity (Baker & Kantrowitz, 1993).…”

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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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Identification and analysis of long-range electrostatic effects in proteins by computer modeling:Aspartate transcarbamylase

et al. 1996

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The conserved residues glutamate-37, aspartate-100, and arginine-269, are important for the structural stabilization of Escherichia coli aspartate transcarbamoylase

Cited by 32 publications

References 59 publications

Stabilization of the R Allosteric Structure of Escherichia coli Aspartate Transcarbamoylase by Disulfide Bond Formation

Stabilization of the R Allosteric Structure of Escherichia coli Aspartate Transcarbamoylase by Disulfide Bond Formation

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

Identification and analysis of long-range electrostatic effects in proteins by computer modeling:Aspartate transcarbamylase

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