Aspartate Transcarbamoylase Part 1 (Molecular Biology)

Aspartate transcarbamoylase (carbamoylphosphate:L-aspartate carbamoyl-transferase, ATCase, EC 2.1.3.2) is a ubiquitous enzyme of pyrimidine biosynthesis in which it catalyzes the first unique step in the de novo synthetic pathway:

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1. The Escherichia coli Paradigm

Although the enzyme exists in a variety of oligomeric and multifunctional complexes in various organisms, the most studied ATCase is that of Escherichia coli. It is a structurally complex, allosterically regulated enzyme containing two catalytic trimers associated with three regulatory dimers, 2(c3):3(^) (Fig. 1). The catalytic polypeptide chain is encoded by thepyrB gene, whereas pyrI encodes the regulatory polypeptide. These genes are preceded by two tandem promoters, designated P1 and P2, and an open reading frame encoding a 44-residue leader polypeptide, pyrL.

Promoter P2 has been identified as the physiologically significant promoter (see Fig. 2). Figure 1. Structure of the intact E. coli ATCase viewed along the 3-fold axis (a) and along the approximate 2-fold axis ( located within the catalytic trimer. The allosteric sites are located within the regulatory dimers. The PDB file 8at1 was us


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Figure 2. Genetic organization of the E. coli pyrLBI operon. The position of the three cistrons on the chromosome is sho positions of three hairpin structures adopted by the messenger RNA. The sequence around the first two is shown below. T pause hairpin, flanked on each side by uridine-rich sequences.

Genetic organization of the E. coli pyrLBI operon. The position of the three cistrons on the chromosome is sho positions of three hairpin structures adopted by the messenger RNA. The sequence around the first two is shown below. T pause hairpin, flanked on each side by uridine-rich sequences.

The pyrLBI operon is located on the linkage map of E. coli at 97 minutes and unlinked to any of the genes or small operons of the other six enzymes involved in pyrimidine biosynthesis. The expression of these genes is noncoordinately regulated by the intracellular levels of uridine or cytidine nucleotides, with the expression of the pyrBI operon negatively regulated over 300-fold. Most of this regulation (50-fold) occurs through a UTP-sensitive attenuation control mechanism, whereas attenuator-independent mechanisms are responsible for approximately a 6.5-fold range of regulation, including a pyrimidine-sensitive transcriptional initiation mechanism, and stringent control by ppGpp (1). According to the current model for attenuation control, transcriptional termination at the pyrBI attenuator (a rho-independent transcriptional terminator) is regulated by the relative rates of transcription and translation within the pyrBI leader region. Low intracellular levels of UTP cause RNA polymerase to pause at the uridine-rich region in the leader transcript (this pausing is enhanced by NusA, a general transcription factor that increases the efficiency of termination). This allows time for a ribosome to initiate translation and catch up to the stalled RNA polymerase before it transcribes the attenuator region. As RNA polymerase eventually makes its way through the attenuator, the adjacent translating ribosome blocks formation of the terminator hairpin. This permits RNA polymerase to read through into the pyrBI structural genes, with translation terminating before thepyrB initiation codon. When intracellular levels of UTP are high, RNA polymerase does not pause during the transcription ofpyrL. Without this pausing, the ribosomes cannot catch up to RNA polymerase before it transcribes the attenuator. This allows formation of the RNA hairpin, thus terminating transcription prior to the structural genes.

In addition to the genetic regulation of pyrLBI, the gene product ATCase also exerts significant control over the rate of pyrimidine biosynthesis (2). This is achieved by allosteric modification of the enzymatic activity in response to substrate concentration (homotropic cooperativity) and the nucleotide end products of both the purine and pyrimidine pathways (heterotropic regulation; see Tables 1 and 2). In the oligomeric complete enzyme, catalysis proceeds by a preferred order mechanism, with carbamoyl phosphate binding before aspartate and N-carbamoyl-L-aspartate leaving before inorganic phosphate. Homotropic cooperativity is induced by aspartate in the presence of a saturating concentration of carbamoyl phosphate and involves a structural transition of the enzyme consistent with a two-state, concerted model. As aspartate is bound, the enzyme shifts from the T-state, characterized by low activity and low affinity for substrates, to the R-state with high activity and high affinity for substrates. The kinetic consequence of this positive cooperativity is a sigmoidal substrate saturation curve (Fig. 3). Heterotropic activation is caused by the purine ATP, whereas the pyrimidine end products CTP and UTP feedback inhibit the enzyme. The pattern of inhibition exhibited by pyrimidine nucleotides is synergistic: CTP inhibits ATCase activity approximately 60% and UTP has minimal effect, while CTP in combination with UTP inhibits ATCase activity >95% (3). CTP, ATP, and UTP bind competitively, with different affinities, to a common allosteric site. CTP binds the most tightly with a pattern consistent with two classes of three sites each, whose dissociation constants differ by a factor of 20 (Kd-CTP: 5 to 20 |M). The binding of ATP follows a pattern similar to that of CTP with two classes of affinity sites, except that ATP binding is an order of magnitude weaker than that of CTP (Kd-ATP: 60 to 100 |M). The binding of UTP appears to be limited to three sites (Kd-UTP: 800 |M), although there may be a second class of sites too weak to be measured. Interestingly, the binding of CTP to three sites appears to enhance the binding of UTP to the remaining three sites almost 100-fold, resulting in a predicted Kd for UTP in the presence of CTP of 10 |M (4). These biochemical binding characteristics are well suited to the physiological requirements of the pathway: Although the intracellular concentration of CTP (500 |M) and UTP (900 |M) is 3- to 6-fold lower than that of ATP (3 to 5 mM), their stronger binding can effectively displace ATP at the allosteric sites of the enzyme.

Figure 3. Substrate saturation profile of the E. coli ATCase. The velocity of the enzyme at varying concentrations of asp presence of no allosteric effectors \ the activator ATP the inhibitor CTP, and both pyrimidine end-products CT.

Substrate saturation profile of the E. coli ATCase. The velocity of the enzyme at varying concentrations of asp presence of no allosteric effectors \ the activator ATP the inhibitor CTP, and both pyrimidine end-products CT

Physiological Significance of Catalytic Characteristics of the E. coli Aspartate Transcarbamoyl

Catalytic Characteristic

Functional Consequence

• Ordered substrate binding tmp5-121

• Places catalytic emphasis on aspartate

• Cooperative aspartate binding

• Allows large changes in catalytic activity with onl of physiological substrate concentrations

• Distinct regulatory and catalytic subunits

• Increased sophistication in the allosteric modulatio activity

• Presence of shared active sites and catalytictrimers

• Facilitates modulation of catalytic activity with sul movements

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• Provides for dramatic differences in aspartate bind modulation of catalytic capacity at physiological lev

Table 2. Physiological Significance of Allosteric Characteristics of the E. coli Aspartat

Allosteric Characteristic

Functional Consequence

• Allosteric inhibition by CTP

• 50-70% inhibition of activity by nucleotide i balance new synthesis with utilization

• Synergistic inhibition by UTP and CTP

• UTP and CTP are both end-products of the b pathway and the synergism provides 90-95% activity

• Competitive allosteric activation by ATP

• ATP competes with CTP and UTP for the sa thus balancing intracellular purine and pyrimi pools

• Competitive allosteric inhibition by

• Allows for displacement of ATP (activation)

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• Negative cooperativity in binding CTP

• Permits modulation of enzymatic activity ov of inhibitor concentration

• Communication of allosteric signals across protein-protein interfaces

• Facilitates modulation of allosteric signal

• Asymmetry of the holoenzyme

• Provides two distinct classes of allosteric bin a single enzyme

Comparison of the X-ray crystallography structures of the enzyme has revealed that each of the catal independently folding structural domains: the carbamoyl phosphate (CP) domain and aspartate (Asp sites are located at the interface between the CP and Asp domains of one catalytic chain and the CP < chain. Likewise, the regulatory chains are composed of two structural domains: the zinc-binding (Zn domain. Differences between the two states of the enzyme have been identified by comparison of the inhibitor (CTP) and the R-state structure binding a bisubstrate analogue [N-(phosphonacetyl)-L-aspa change in quaternary structure involves substantial conformational rearrangements as the catalytic tri mutually reorient 10° around the 3-fold axis, while each regulatory dimer rotates 15° around the 2-fo binding, the two domains of each catalytic chain (Asp and CP) undergo domain closure, whereas the chain (Alio and Zn) undergo domain separation. Site-directed mutagenesis studies have shown that c domains is important for the formation of the high-affinity, high-activity R-state, which is required t< conformation needed for catalysis, and for homotropic cooperativity.

Figure 4. Domain organization of the E. coli ATCase illustrated with one catalytic chain (C1) and its associated regulal composed of two independently folding domains: the aspartate-binding (Asp) domain and carbamoyl phosphate-binding regulatory chain is composed of the zinc-binding (Zn) domain and nucleotide-binding (allosteric) domain. This R1:C1 u MOLSCRIPT programs.

Domain organization of the E. coli ATCase illustrated with one catalytic chain (C1) and its associated regulal composed of two independently folding domains: the aspartate-binding (Asp) domain and carbamoyl phosphate-binding regulatory chain is composed of the zinc-binding (Zn) domain and nucleotide-binding (allosteric) domain. This R1:C1 u MOLSCRIPT programs.

In addition to these studies, well over 100 site- and region-specific mutations have been created in th studies, along with the many structural and biochemical analyses, have contributed to our understand active-site and allosteric site locations, identifying interactions important to the stabilization of the T catalytically significant residues. More recently, site-directed mutagenesis studies have been directed mechanism. Mutations have been created that can effectively separate the homotropic from the heter from inhibition, and CTP inhibition from CTP + UTP synergism. Although the mechanism of alloste current mutagenesis and structural studies are attempting to identify and differentiate between possib discreet pathways for each nucleotide signal, (2) complex and multiple interlocking pathways, or (3) energy changes.

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