E. coliAspartate Transcarbamoylase

(ATCase)

Wang et al. (2005) Proc Natl Acad Sci USA 102, 8881-6.

Macol et al. (2001) Nat Struct Biol 8, 423-6.

Helmstaedt et al. (2001) Micr Mol Biol Rev 65, 404-21.

E. coli ATCase

Carbamoyl-phosphate (CP) + L-aspartate

--> N-carbamoyl-L-aspartate

•First enzyme unique to pyrimidine biosynthesis.

•Key enzyme for regulating pyrimidine, purine, and argingine biosynthesis.

Homotropic ligand: L-aspartate (positive cooperativity)

Heterotropic ligands: ATP (allosteric activator)

UTP and CTP (allosteric inhibitor)

Mammalian ATCase

• Component of CAD multi-enzyme complexCP synthetaseATCasedihydroortase

• Target for antiproliferative drug development.

• E. coli ATCase and ATCase portion of CAD are 44% conserved.

• All residues known to be involved in substrate binding and catalysis are conserved.

E. coli ATCaseOrdered sequential BiBi kinetic mechanism

CP Asp PiNCA

E E.CP [ ] E.Pi E

Tetrahedral intermediate

E. coli ATCase

•6 x 34 kDa chains grouped into 2 trimeric catalytic subunits

•6 x 17 kDa chains grouped into 3 dimeric regulatory subunits

•3 active sites in each catalytic subunit (trimer) are shared across the interface between adjacent chains.

•Each catalytic chain has 2 domains:

CP (aa 1-135 and 292-310)

Asp (aa 136-291)

•Regulatory subunits contain binding sites for heterotropic activator (ATP) and heterotropic inhibitors (CTP & UTP).

•Each regulatory chain has 2 domains:

AL (aa 1-100)

Zn (aa 101-153)

E. coli ATCase

Holoenzyme viewed along 3-fold axis. Binding mode of bisubstrate analog PALA to active site.

The active sites contain aa from the CP and Asp domains of one C-chain and from the CP domain of an adjacent C-chain.

E. coli ATCase

Holoenzyme viewed along 3-fold axis. Binding mode of bisubstrate analog PALA to active site.

The allosteric sites are located at the distal ends of the R-chains, 60 Å away from the nearest active site, and bind each effector.

E. coli ATCase

Holoenzyme viewed along 3-fold axis. Binding mode of bisubstrate analog PALA to active site.

Assembly into the holoenzyme yields extensive interfaces between the C-chains within each catalytic trimer (e.g. C1-C2) and in opposed trimers (e.g. C1-C4). The C1-C4 (and symmetry related) interfaces are present in the T state but not in the R state.

T state

R state

E. coli ATCase

Holoenzyme viewed along 3-fold axis. Binding mode of bisubstrate analog PALA to active site.

Assembly into the holoenzyme also yields extensive interfaces between the R-chains within each regulatory dimer (e.g. R1-R6) and between C- and R-chains (e.g. C1-R1 and C1-R4).

Substrate-induced Domain Closure in E. coli ATCase-- triggers quaternary conformational change that results in homotropic cooperativity.

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• Triggered by ordered binding of substrates.

• Induces dramatic quaternary structural change, from tense (T) to relaxed (R) form.

11 Å elongation of molecule.

rotations of C and R subunits.

• Promotes catalysis by correctly orienting active site aa side chains for high-affinity substrate binding and catalysis.

• Because of structural constraints, the closure of the domains of the catalytic chains cannot occur without the global quaternary conformational change, which allows domain closure without steric interference.

• The global conformational change can be triggered by the binding of a single molecule of bisubstrate analog to just 1 of the 6 active sites.

Structural basis for ordered substrate binding and cooperativity in E. coli ATCase:

Copyright ©2005 by the National Academy of Sciences

Wang, Jie et al. (2005) Proc. Natl. Acad. Sci. USA 102, 8881-8886

Fig. 1. Comparison of the x-ray structures of ATCase in the absence and presence of CP

1 C-chain of ATCase along with the 80’s loop of the adjacent C-chain

Overlay of CP binding site in absence and prescence of CP

Active site without CP

Active site with CP

Electrostatic surface potential without CP

Electrostatic surface potential with CP

Copyright ©2005 by the National Academy of Sciences

Wang, Jie et al. (2005) Proc. Natl. Acad. Sci. USA 102, 8881-8886

Fig. 2. Stereoview of a portion of the active site of ATCase

CP and Asp would bind to the same site of unliganded ATCase

By binding first, CP induces a local conformational change that creates the correct site for Asp binding.

Asp is positioned correctly for nucleophilic attack.

CP-induced conf. change also renders active site more electropositive-- lowers pKa of Asp -NH2 promoting catalysis.

CP binding weakens interactions that stabilize the T state.

Asp binding induces allosteric conformational change.

Asp binding renders active site more electronegative.

This causes positively charged residues in the 80’s and 240’s loops to reposition closer to the substrates.

These backbone motions correlate with domain closure leading to the R state.

The link between catalysis and homotropic cooperativity:

Movement of 80’s and 240’s loop towards substrates triggered by Asp binding forces substrates closer together, lowering energy of activation.

The same movement further weakens intersubunit interactions that specifically stabilize the T state, triggering the global quaternary conformational change.

The 240’s loop cannot attain the final, domain-closed conformation without an expansion of the enzyme along the 3-fold axis, which allows the 240’s loops from the upper and lower catalytic trimers to slide past each other.

The link between domain closure and homotropic cooperativity.

Ordered substrate binding is explained by the induced-fit conformational change upon CP binding.

This change dramatically transforms the electrostatics of the active site, creating the binding site for Asp.

Asp binding changes th electrostatics once more, causing a second induced-fit: domain closure.

Domain closure facilitates catalysis and induces the quaternary conformational change from T --> R state, resulting in homotropic cooperativity.