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aspartate transcarbamoylase

aspartate transcarbamoylase (ATCase)

The "hemoglobin of enzymology", ATCase is the most extensively studied example of an allosteric enzyme. The enzyme from E. coli is a dodecamer consisting of six catalytic (c) subunits and six regulatory (r) subunits, for a composition of c6r6. ATCase [EC] catalyzes the first step in pyrimidine biosynthesis, namely the addition of carbamoyl phosphate to L-aspartate to form N-carbamoylaspartate and phosphate. The following step in the pathway is cyclization of N-carbamoylaspartate to dihydroorotate.
Diagram of ATCase reaction, showing substrate and product structures
The reaction likely proceeds through a tetrahedral intermediate after the α-amino group of aspartate attacks the carbonyl carbon of carbamoyl phosphate, and prior to the expulsion of the orthophosphate leaving group. Note that CbmP is a fairly high-energy phosphoric anhydride, making this reaction energetically favorable (energy is required to synthesize CbmP).
Diagram of ATCase reaction mechanism, showing strucutres of substrates, products, and intermediate
  Structural diagram of PALA, a bisubstrate analog inhibitor of ATCase   An extremely useful inhibitor of ATCase known as PALA [N-(phosphonacetyl)-L-aspartate] is a bisubstrate analog., PALA is a non-hydrolyzable phosphonate compound which is otherwise similar to CbmP + aspartate, except for a missing amino group. PALA has a Kd of about 10 nM for ATCase, meaning it binds the enzyme quite strongly. Another inhibitor of ATCase is the dicarboxylic acid succinate, and the combination of CbmP + succinate is again similar to CbmP + aspartate, except it is missing the α-amino group of aspartate.
Gerhart and Pardee found in 1962 that ATCase displays sigmoidal kinetics, meaning the reaction velocity vs. substrate concentration curve has a sigmoidal or S-shaped form. The enzyme shows a strong preference for binding of CbmP first. Once CbmP is bound, the affinity of the enzyme for aspartate is increased markedly, and aspartate shows a positive cooperative effect on catalysis.

X-ray crystallographic studies of ATCase

The first structure of ATCase, that of the unliganded holoenzyme (i.e. the c6r6 heterododecamer), was determined in 1982 by William Lipscomb and associates (2.6 Å resolution). Various other structures of complexes of ATCase and ligands followed. The structure of the unliganded ATCase holoenzyme showed that the dodecamer was constructed of two catalytic trimers (c3) separated along an axis corresponding to a three-fold symmetry axis, and bridged by three regulatory dimers (r2).

ATCase structure (thumbnail)  

Selected ATCase structures:

6at1.pdb - unliganded ATCase
1rab.pdb - T state (holoenzyme) complex with CTP
1d09.pdb - PALA-ligated holoenzyme at 2.1 Å
3csu.pdb - unliganded catalytic trimer
1ekx.pdb - PALA-liganded catalytic trimer

Click on any of the links above or go to the PDB (Protein Data Bank) home page and search for ATCase or aspartate transcarbamoylase or type in any of the 4-character codes above to go to the entry for that structure.

Structural changes induced by substrate analog (PALA) binding

An important early observation made in the studies of ATCase was that binding of substrate analogs correlated with an increase in the hydrodynamic volume of the enzyme, as measured by the rate of sedimentation in an analytical ultracentrifuge. This was evidence of a change in the enzyme's quaternary structure induced by substrate binding. The structures of ATCase determined by X-ray crystallography revealed striking differences between the unliganded and PALA-saturated holoenzyme. Compared to the unliganded holoenzyme, the catalytic trimers of the PALA-ligated form are further separated from one another, as allowed by a rotation of the regulatory dimers (see figure below). Furthermore, changes occurred in the tertiary structures of the c chains. The two domains that form the active site between them approached each other more closely, a hinge-like motion permitted by the loss of contacts between the two catalytic trimers as these become further separated. When correlated with the dependence of holoenzyme activity with substrate concentration, the observed expansion of ATCase was interpreted as corresponding to a change from a low-affinity, low-activity state of the holoenzyme to a high-affinity, high-activity form - in short, a T to R transition. The fact that substoichiometric amounts of PALA induced the hydrodynamic expansion and stimulated holoenzyme activity suggested that the symmetry, or MWC (Monod-Wyman-Changeux) model could be applied to the allosteric behavior of ATCase. The tertiary and quaternary changes in ATCase are tightly coupled, so the two-state model works especially well in this case.

Ribbon representations of ATCase holoenzyme (l) with CTP, (r) with PALA

Above: The structures, represented in ribbon form, of ATCase holoenzyme with CTP bound (left), representing what has been interpreted as the lower activity T state, and with PALA bound (right), interpreted as the higher activity R state. In both cases, the catalytic trimers are show in cyan, and the regulatory dimers are colored magenta. Note that the expansion of the PALA-bound structure results in the nearly complete separation of catalytic trimers, as opposed to the structure on the left in which there are extensive contacts between the trimers. The ligands (CTP and PALA, respectively) are shown as sticks (carbon atoms are colored yellow). CTP binds stoichiometrically to the regulatory subunits, and is readily visible in the structure at left. PALA binds to each of the three active sites formed by each catalytic trimer. The figure on the left is based on 1rab.pdb - and the figure on the right is based on 1d09.pdb, and the images generated using PyMOL (Ref.5).


  1. Beernink PT, Endrizzi JA, Alber T & Schachman HK. (1999). Assessment of the allosteric mechanism of aspartate transcarbamoylase based on the crystalline structure of the unregulated catalytic subunit. Proc Natl Acad Sci USA 96: 5388-5393.
  2. Endrizzi JA, Beernink PT, Alber T & Schachman HK. (2000). Binding of bisubstrate analog promotes large structural changes in the unregulated catalytic trimer of aspartate transcarbamoylase: Implications for allosteric regulation. Proc Natl Acad Sci USA 97: 5077-5082.
  3. Kantrowitz ER & Lipscomb WN. (1988). Escherichia coli Aspartate Transcarbamylase: The Relation Between Structure and Function. Science 241: 669-674.
  4. Schachman HK. (2000). Still looking for the ivory tower. Annu Rev Biochem 69: 1-29.
  5. The PyMOL Molecular Graphics System, Version Schrödinger, LLC.


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© 2014 Jeff D Cronk, Gonzaga University