Calorimetric analysis of aspartate transcarbamylase from Escherichia coli. Binding of cytosine 5'-triphosphate and adenosine 5'-triphosphate

Nm, Allewell; Friedland, Joan; Niekamp, K

doi:10.1021/bi00673a005

Cited by 37 publications

(27 citation statements)

References 40 publications

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“…The half-saturating concentration for ATP activation was 0.8 mM, whereas CTP-induced inhibition required levels that were decreased by a factor of -5 (0.15 mM). This weaker binding of ATP has been described by others (19,20) and would explain the dominance of CTP inhibition over ATP activation. The nature of this competitive advantage for CTP can be described in a slightly different manner by evaluating competitive saturation curves (data not shown).…”

Section: Resultssupporting

confidence: 68%

Section: Introductionmentioning

confidence: 99%

“…These allosteric controls have been promoted for their inherent logic in balancing endogenous purine and pyrimidine nucleotide pools; however, there has always been a quantitative paradox relative to the incomplete inhibition by CTP (10, 14, 15). In addition, it appears that there is discrete heterogeneity in CTP binding as half of the allosteric sites are capable of providing for high-affinity binding of CTP whereas three additional, lowaffinity sites have been identified in some studies (16)(17)(18)(19).A potential structural basis for this "half-site reactivity" has been provided by recent studies of Lipscomb and coworkers (20) in which a molecular asymmetry has been observed across the "two-fold axis of symmetry" of the CTP-liganded enzyme. In the presence of saturating concentrations of CTP, one of the allosteric sites of each dimer is fully occupied, whereas the second has an occupancy factor of <0.4.…”

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confidence: 99%

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In the presence of CTP, UTP becomes an allosteric inhibitor of aspartate transcarbamoylase.

Wild

Loughrey-Chen

Corder

1989

Proc. Natl. Acad. Sci. U.S.A.

125

117

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The allosteric control of aspartate transcarbamoylase (ATCase, EC 2.1.3.2) of Escherichia coli involves feedback inhibition by both CTP and UTP rather than just CTP alone. It has been known that CTP functions as a heterotropic inhibitor of catalysis; however, the inhibition by CTP alone is incomplete (50-70% at various aspartate concentrations) and there is only a partial occupancy of the allosteric binding sites by CTP at saturating concentrations. The logic of these allosteric characteristics can now be understood in that UTP is a synergistic inhibitor of ATCase in the presence of CTP even though UTP has no independent effect at pH 7.0. When saturating concentrations of CTP are present, the concentration of substrate required for half-maximal activity (So.s) of the native holoenzyme for aspartate increases from 5 to 11 mM. When CTP and UTP are both present, the aspartate requirement increases further (S0.5 = 17 mM). At aspartate concentrations <5 mM, the heterotropic inhibition of ATCase is 90-95% in the presence of both pyrimidine nucleotides. UTP does enhance the binding of CTP to the holoenzyme but the number of tight binding sites does not change (n = 3). Biol. 196, have described a structural asymmetry across the molecular two-fold axis that is consistent with these CTP/UTP interactions. The synergistic inhibition of ATCase by both CTP and UTP provides a satisfying logic for ensuring a balance of endogenous pyrimidine nucleotide pools.The aspartate transcarbamoylase (ATCase, EC 2.1.3.2) of Escherichia coli provides a classic example of an enzyme subject to allosteric control by the end product of its biosynthetic pathway (1, 2). The holoenzyme of ATCase is composed of two separable catalytic trimers (c3) and three regulatory dimers (r2) that interact through a variety of specific protein-protein interfaces (3, 4). These interactions provide for specific conformational transitions (4-7) of the holoenzyme, 2(c3):3(r2), which result in changes in the catalytic rates and ligand affinities (8-10). Upon binding of substrates and/or substrate analogues the enzyme appears to make a concerted transition from an inactive "T conformation" (condensed form of the enzyme) to an expanded, more active "R conformation" (5-7). CTP affects catalysis by increasing the concentration of aspartate required to produce maximal activity. In contrast, ATP decreases the substrate requirement without altering the Vmax of the enzyme (1, 2).Both nucleotide effectors have been shown to competitively bind at the same allosteric sites even though they induce different heterotropic effects on catalysis (11)(12)(13)(14). Other pyrimidine nucleotides, specifically UTP and TTP, do not have any effect. GTP does promote a significant inhibitory effect under some conditions (25-30%) but less than that of CTP (50-70%). 8-Bromoguanosine 5'-triphosphate, as well as gadolinium complexes of ATP, CTP, and GTP, binds to the regulatory subunits, whereas UTP does not appear to bind to the native enzyme (11-13). These allosteric controls have bee...

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Section: Resultssupporting

confidence: 68%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

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In the presence of CTP, UTP becomes an allosteric inhibitor of aspartate transcarbamoylase.

Wild

Loughrey-Chen

Corder

1989

Proc. Natl. Acad. Sci. U.S.A.

125

117

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“…This study also suggested that the pH‐dependent shift in the Asp saturation curve is largely because of changes involving intersubunit salt bridges. Finally, a detailed study of the Bohr effect in ATCase was performed using calorimetry, potentiometry, and spectrophotometry to analyze the coupling between the binding of protons and substrate analogues 15, 17. This work identified two residues with p K a values of 6.3 and a third residue with a pK a of 9.1 that play a critical role in the Bohr effect of ATCase 18…”

Section: Introductionmentioning

confidence: 99%

The first high pH structure of Escherichia coli aspartate transcarbamoylase

2008

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The activity and cooperativity of Escherichia coli aspartate transcarbamoylase (ATCase) vary as a function of pH, with a maximum of both parameters at approximately pH 8.3. Here we report the first X-ray structure of unliganded ATCase at pH 8.5, to establish a structural basis for the observed Bohr effect. The overall conformation of the active site at pH 8.5 more closely resembles the active site of the enzyme in the R-state structure than other T-state structures. In the structure of the enzyme at pH 8.5 the 80's loop is closer to its position in R-state structures. A unique electropositive channel, comprised of residues from the 50's region, is observed in this structure, with Arg54 positioned in the center of the channel. The planar angle between the carbamoyl phosphate and aspartate domains of the catalytic chain is more open at pH 8.5 than in ATCase structures determined at lower pH values. The structure of the enzyme at pH 8.5 also exhibits lengthening of a number of interactions in the interface between the catalytic and regulatory chains, whereas a number of interactions between the two catalytic trimers are shortened. These alterations in the interface between the upper and lower trimers may directly shift the al-losteric equilibrium and thus the coopera-tivity of the enzyme. Alterations in the elec-tropositive environment of the active site and alterations in the position of the cata-lytic chain domains may be responsible for the enhanced activity of the enzyme at pH 8.5.

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“…pK values of groups involved in binding and/or catalysis have been derived from the pH dependence of the kinetic parameters of c 3 (Leger and Hervé, 1988;Turnbull et al, 1992), and a model involving three active site residues has been proposed (Turnbull et al, 1992). Allewell et al (1975) showed that nucleoside triphosphate binding is linked to proton binding, and in a series of subsequent papers they defined linkages between proton binding and binding of substrates, competitive inhibitors, and nucleotides and the assembly reaction (Knier and Allewell, 1978;Allewell et al, 1979;Burz and Allewell, 1982;McCarthy and Allewell, 1983). Computer modeling indicates that these linkages result from large changes in the pK a values of groups widely distributed throughout the molecule (Glackin et al, 1989(Glackin et al, , 1991.…”

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confidence: 99%

Effects of Assembly and Mutations Outside the Active Site on the Functional pH Dependence of Escherichia coli Aspartate Transcarbamylase

Yuan

LiCata²,

Allewell³

1996

Journal of Biological Chemistry

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The tyrosines at positions 165 and 240 are part of a cluster of interactions that links the catalytic subunits in the T state (the c1:c4 interface) and which is disrupted in the T 3 R transition. The effects of mutating the two Tyr residues are quite different: Y240F has higher than wild-type activity and affinity over the entire pH range, while Y165F has activity and affinity an order of magnitude lower than wild-type. Removal of the regulatory subunits from Y165F increases activity and affinity and restores the pH dependence of the wildtype catalytic subunit. Like Y165F, E50A has low activity and affinity over the entire pH range. Linkage analysis indicates that there is long range energetic coupling among the active site, the c:r subunit interfaces, and residue Y165. The substantial quantitative difference between Y165F and Y240F, both of which are at the c1:c4 interface about 14 -16 Å from the closest active site, demonstrates specific path dependence, as opposed to general distance dependence, of interactions between this interface and the active site.Escherichia coli aspartate transcarbamylase (EC 2.1.3.2) was one of the first systems exploited to study intramolecular signal transduction (Yates and Pardee, 1956) and remains an important system for analyzing molecular mechanisms of recognition, communication, and regulation. This enzyme catalyzes the first committed step in pyrimidine biosynthesis, formation of N-carbamyl-L-Asp from carbamyl phosphate and L-aspartate. Aspartate transcarbamylase binds L-Asp cooperatively, and its enzymatic activity is allosterically regulated by nucleotide triphosphates (Yates and Pardee, 1956; Pardee, 1962, 1963;Wild et al., 1989). Recent reviews include Allewell (1989), Hervé (1989), Kantrowitz and Lipscomb (1990), Wild and Wales (1990), and Lipscomb (1992.The protein is a dodecamer consisting of six catalytic (c) 1 chains and six regulatory (r) chains organized as two catalytic trimers (c 3 ) and three regulatory dimers (r 2 ). Binding of substrates and substrate analogs induces a T 3 R transition in which the holoenzyme expands by 12 Å along its 3-fold axis. The switch to the high affinity R structure eliminates contacts between c 3 subunits (the C1:C4 interface), one set of c 3 -r 2 contacts and domain interactions in the r chain but simultaneously strengthens a second set of c 3 -r 2 contacts, interchain interactions in c 3 and interdomain interactions in c chains.Critical features of this transition include large movements of the 80s and 240s loops in the c chains and closure of the c chain domains (see Fig. 1). Because both the substrates of aspartate transcarbamylase and its regulatory nucleotides have several negative charges, electrostatic effects might be expected to figure heavily in both catalysis and the allosteric mechanism. A number of studies have substantiated this expectation. Gerhart and Pardee (1964) showed that the pH optimum for catalytic activity shifts from 7 at low aspartate concentrations to 8.3 at high aspartate concentrations. These pH optima a...

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Calorimetric analysis of aspartate transcarbamylase from Escherichia coli. Binding of cytosine 5'-triphosphate and adenosine 5'-triphosphate

Cited by 37 publications

References 40 publications

In the presence of CTP, UTP becomes an allosteric inhibitor of aspartate transcarbamoylase.

In the presence of CTP, UTP becomes an allosteric inhibitor of aspartate transcarbamoylase.

The first high pH structure of Escherichia coli aspartate transcarbamoylase

Effects of Assembly and Mutations Outside the Active Site on the Functional pH Dependence of Escherichia coli Aspartate Transcarbamylase

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