Kinetic mechanism of native Escherichia coli aspartate transcarbamylase

Hsuanyu, Yuchiong; Wedler, Frederick C.

doi:10.1016/0003-9861(87)90498-x

Cited by 59 publications

(64 citation statements)

References 32 publications

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“…The maximal activity of the C47A/A241C holoenzyme was greater under non-reducing versus reducing conditions (25.6 versus 21 mmol⅐h Ϫ1 ⅐mg Ϫ1 respectively), suggesting that the switch from the T to R state is somewhat rate-limiting in the holoenzyme. This is supported by work done on the wildtype enzyme and D236A, another enzyme with enhanced maximal activity, which suggested that domain closure or "compression," the final step in the T to R transition, is rate-limiting (37,38). There was a significant difference in the nucleotide saturation curves under reducing and non-reducing conditions corresponding to a wild-type-like and R state enzyme, respectively.…”

Section: Discussionmentioning

confidence: 80%

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: Discussionmentioning

confidence: 80%

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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“…This enzyme (Mr 305646), composed of two catalytic trimers and three regulatory dimers, catalyzes the first reaction of the pyrimidine biosynthetic pathway, the carbamylation of the amino group of aspartate by carbamylphosphate (Hsuanyu & Wedler, 1987;Reichard & Hanshoff, 1956). ATCase, whose activity is synergistically feedback-inhibited by CTP and UTP, the end products of the pyrimidine pathway and stimulated by ATP, exhibits homotropic cooperativity between the catalytic sites for the binding of the substrate aspartate (Bethell et al, 1968;Gerhart & Pardee, 1962).…”

Section: Introductionmentioning

confidence: 99%

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

Tauc¹,

Vachette²

1997

J Appl Cryst

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The allosteric enzyme aspartate transcarbamylase from Escherichia coli (ATCase) displays regulatory properties that involve various conformational changes, including a large quaternary structure rearrangement. This entails a major change in its solution X-ray scattering curve upon binding substrate analogues, thereby providing a direct means to monitor the amount of different quatemary structures present in solution. Scattering curves in the presence of variable concentrations of several such substrate analogues were recorded using an area detector. Data were analyzed by singular-value decomposition without any prior assumption as to the number of quaternary structure states. They can all be accounted for with only two states. The structural transition appears to be concerted, a conclusion whose validity has been extended to higher resolution and to other combinations of ligands than previously studied using a linear detector. The titration curve with the bisubstrate analogue N-(phosphonacetyl)-L-aspartate (PALA) alone is identical to that previously obtained, while that in the additional presence of carbamyl phosphate (CP) shows a clear R shift. However, the scattering pattern of the ATCase-CP complex is shown to be different from the T pattern of the unligated enzyme, though not a linear combination of the Tand R patterns. Therefore, we conclude that CP acts by modifying the quaternary structure of the unligated enzyme to a T-like structure, a structure more easily converted to the R conformation by subsequent addition of PALA. AbbreviationsATCase = aspartate transcarbamylase (or carbamoyltransferase) from E. coli (EC 2.1.3.2.); CP = carbamyl phosphate; PALA = N-(phosphonacetyl)-L-aspartate; T = quaternary structure state in the absence of ligand; R = quaternary structure state in the presence of saturating concentrations of PALA or substrates; SVD = singularvector decomposition; SAXS = small-angle X-ray scattering.

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“…from Escherichia coli catalyzes the first step of pyrimidine biosynthesis, the condensation of carbamoyl phosphate and L-aspartate to form Ncarbamoyl-L-aspartate and phosphate. The reaction mechanism is "preferred order" with carbamoyl phosphate binding before aspartate, and N-carbamoylaspartate leaving before phosphate (Hsuanyu & Wedler, 1987). The E. coli enzyme is endowed with …”

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confidence: 99%

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

Tauc¹,

Keiser²,

Kantrowitz³

et al. 1994

Protein Science

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Glu-50 of aspartate transcarbamoylase from Escherichia coli forms a set of interdomain bridging interactions between the 2 domains of the catalytic chain; these interactions are critical for stabilization of the high-activity highaffinity form of the enzyme. The mutant enzyme with an alanine substituted for Glu-50 (Glu-50 + Ala) exhibits significantly reduced activity, little cooperativity, and altered regulatory behavior (Newton CJ, Kantrowitz ER, 1990, Biochemistry 29:1444-1451). A study of the structural consequences of replacing Glu-50 by alanine using solution X-ray scattering is reported here. Correspondingly, in the absence of substrates, the mutant enzyme is in the same, so-called T quaternary conformation as is the wild-type enzyme. In the presence of a saturating concentration of the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA), the mutant enzyme is in the same, so-called R quaternary conformation as the wild-type enzyme. However, the Glu-50 + Ala enzyme differs from the wild-type enzyme, in that its scattering pattern is hardly altered by a combination of carbamoyl phosphate and succinate. Addition of ATP under these conditions does result in a slight shift toward the R structure. Steadystate kinetic studies indicate that, in contrast to the wild-type enzyme, the Glu-50 + Ala enzyme is activated by PALA at saturating concentrations of carbamoyl phosphate and aspartate, and that PALA increases the affinity of the mutant enzyme for aspartate. These data suggest that the enzyme does not undergo the normal T to R transition upon binding of the physiological substrates and verifies the previous suggestion that the interdomain bridging interactions involving Glu-50 are critical for the creation of the high-activity, high-affinity R state of the enzyme.Keywords: aspartate carbamoyltransferase; quaternary structure; single amino acid mutation; solution X-ray scattering Aspartate transcarbamoylase (EC 2.1.3.2.) from Escherichia coli catalyzes the first step of pyrimidine biosynthesis, the condensation of carbamoyl phosphate and L-aspartate to form Ncarbamoyl-L-aspartate and phosphate. The reaction mechanism is "preferred order" with carbamoyl phosphate binding before aspartate, and N-carbamoylaspartate leaving before phosphate (Hsuanyu & Wedler, 1987). The E. coli enzyme is endowed with

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Kinetic mechanism of native Escherichia coli aspartate transcarbamylase

Cited by 59 publications

References 32 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

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

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

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