The N-Terminus of the Regulatory Chain of <i>Escherichia coli</i> Aspartate Transcarbamoylase Is Important for both Nucleotide Binding and Heterotropic Effects

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Stabilization of the T and R allosteric states of Escherichia coli aspartate transcarbamoylase is governed by specific intra-and interchain interactions. The six interchain interactions between Glu-239 in one catalytic chain of one catalytic trimer with both Lys-164 and Tyr-165 of a different catalytic chain in the other catalytic trimer have been shown to be involved in the stabilization of the T state. In this study a series of hybrid versions of aspartate transcarbamoylase was studied to determine the minimum number of these Glu-239 interactions necessary to maintain homotropic cooperativity and the T allosteric state. Hybrids with zero, one, and two Glu-239 stabilizing interactions do not exhibit cooperativity, whereas the hybrids with three or more Glu-239 stabilizing interactions exhibit cooperativity. The hybrid enzymes with one or more of the Glu-239 stabilizing interactions also exhibit heterotropic interactions. Two hybrids with three Glu-239 stabilizing interactions, in different geometric relationships, had identical properties. From this and previous studies, it is concluded that the 239 stabilizing interactions play a critical role in the manifestation of homotropic cooperativity in aspartate transcarbamoylase by the stabilization of the T state of the enzyme. As substrate binding energy is utilized, more and more of the T state stabilizing interactions are relaxed, and finally the enzyme shifts to the R state. In the case of the Glu-239 stabilizing interactions more than three of the interactions must be broken before the enzyme shifts to the R state. The interactions between the catalytic and regulatory chains and between the two catalytic trimers of aspartate transcarbamoylase provide a global set of interlocking interactions that stabilize the T and R states of the enzyme. The substrate-induced local conformational changes observed in the structure of the isolated catalytic subunit drive the quaternary T to R transition of aspartate transcarbamoylase and functionally induced homotropic cooperativity.Allosteric regulation is modulated by molecular transitions within a polymeric enzyme between a low activity low affinity T state and a high activity high affinity R state (1). The two states may differ on the quaternary level due to differences in the relative positions of subunits with respect to each other. Interface contacts may undergo significant changes during the T to R state transition, and these contacts often contribute to the relative stabilization of the two states of the enzyme.Escherichia coli aspartate transcarbamoylase (EC 2.1.3.2), which catalyzes the committed step of pyrimidine biosynthesis, the condensation of carbamoyl phosphate and L-aspartate to form N-carbamoyl-L-aspartate and inorganic phosphate (2), has become a model system for the study of allosteric regulation. The enzyme shows homotropic cooperativity for the substrate L-aspartate and is heterotropically regulated by ATP, CTP (2), and UTP in the presence of CTP (3). The holoenzyme from E. coli is a dodecamer composed o...

Section: Methodsmentioning

confidence: 99%

The Contribution of Individual Interchain Interactions to the Stabilization of the T and R States of Escherichia coliAspartate Transcarbamoylase

Sakash

Kantrowitz²

2000

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“…Formation and Purification of Reconstituted Holoenzymes-Equal amounts of purified R105A-C or R54A-C catalytic subunit and AT-C catalytic subunit were mixed with excess regulatory subunit and dialyzed overnight against 50 mM Tris acetate buffer, pH 8.3, 2 mM 2-mercaptoethanol, and 0.1 mM zinc acetate (17). The mixture was examined by nondenaturing PAGE to confirm the existence of the three holoenzyme species, (R105A-C) 2 R 3 , (R105A-C)(AT-C)R 3 , and (AT-C) 2 R 3 or (R54A-C) 2 R 3 , (R54A-C)(AT-C)R 3 , and (AT-C) 2 R 3 .…”

Section: Methodsmentioning

confidence: 99%

Importance of Domain Closure for the Catalysis and Regulation ofEscherichia coli Aspartate Transcarbamoylase

Macol¹,

Tsuruta²,

Kantrowitz³

2002

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Two hybrid versions of Escherichia coli aspartate transcarbamoylase were studied to determine the influence of domain closure on the homotropic and heterotropic properties of the enzyme. Each hybrid holoenzyme had one wild-type and one inactive catalytic subunit. In the first case the inactive catalytic subunit had Arg-54 replaced by alanine. The holoenzyme with this mutation in all six catalytic chains exhibits a 17,000-fold reduction in activity, no loss in substrate affinity, and an R state structurally identical to that of the wild-type enzyme. In the second case, the inactive catalytic subunit had Arg-105 replaced by alanine. The holoenzyme with this mutation in all six catalytic chains exhibits a 1,100-fold reduction in activity, substantial loss in substrate affinity, and loss of the ability to be converted to the R state. Thus, the R54A substitution results in a holoenzyme that can undergo closure of the catalytic chain domains to form the high activity, high affinity active site and to undergo the allosteric transition, whereas the R105A substitution results in a holoenzyme that can neither undergo domain closure nor the allosteric transition. The hybrid holoenzyme with one wild-type and one R54A catalytic subunit exhibited the same maximal velocity per active site as the wild-type holoenzyme, reduced cooperativity, and normal heterotropic interactions. The hybrid with one wild-type and one R105A catalytic subunit exhibited significantly reduced maximal velocity per active site as compared with the wild-type holoenzyme, reduced cooperativity, and substantially reduced heterotropic interactions. Small angle x-ray scattered was used to verify that the R105A-containing hybrid could attain an R state structure. These results indicate the global nature of the conformational changes associated with the allosteric transition in the enzyme. If one catalytic subunit cannot undergo domain closure to create the active sites, then the entire molecule cannot attain the high activity, high activity R state.The homotropic cooperativity in Escherichia coli aspartate transcarbamoylase (EC 2.1.3.2) is directly related to the ability of the substrates to induce a structural and functional transition of the enzyme from a low activity low affinity T state to a high activity, high affinity R state (1). Structurally the conversion of the enzyme from the T to the R state involves an elongation of the enzyme by 11 Å along the molecular 3-fold axis along with a twist of the two catalytic subunits of about 5°in opposite directions (2). This structural transition also results in a simultaneous rotation of each regulatory dimer about its own 2-fold axis. In addition to these quaternary structural changes, the T to R transition involves structural alterations on the tertiary level. For example, during the T to R transition each catalytic chain undergoes a domain closure in which the aspartate domain moves 3 Å toward the carbamoyl phosphate domain, resulting in a major reorganization of the interdomain interface. In addition to this ...

“…Holoenzymes-Equal amounts of purified E50A-C catalytic subunit and AT-C catalytic subunit were mixed with excess regulatory subunit and then dialyzed overnight against 50 mM Tris acetate buffer, pH 8.3, 2 mM 2-mercaptoethanol, and 0.1 mM zinc acetate (23). The mixture was checked by nondenaturing PAGE to confirm the existence of the three holoenzyme species, (E50A-C) 2 R 3 (reconstituted mutant holoenzyme with Ala substituted in place of Glu 50 in the catalytic chain), (E50A-C)(AT-C)R 3 (mutant hybrid holoenzyme in which one catalytic subunit has Ala substituted in place of Glu 50 in each catalytic chain and the other catalytic subunit has 6 aspartate residues added to the C terminus of each catalytic chain), and (AT-C) 2 R 3 (the reconstituted mutant holoenzyme in which 6 aspartate residues have been added to the C terminus of each catalytic chain).…”

Section: Methodsmentioning

confidence: 99%

Domain Bridging Interactions

Sakash¹,

Williams²,

Tsuruta³

et al. 2001

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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...