Intersubunit communication in the dihydroorotase-aspartate transcarbamoylase complex of Aquifex aeolicus

Abstract

Aspartate transcarbamoylase and dihydroorotase, enzymes that catalyze the second and third step in de novo pyrimidine biosynthesis, are associated in dodecameric complexes in Aquifex aeolicus and many other organisms. The architecture of the dodecamer is ideally suited to channel the intermediate, carbamoyl aspartate from its site of synthesis on the ATC subunit to the active site of DHO, which catalyzes the next step in the pathway, because both reactions occur within a large, internal solvent-filled cavity. Channeling usually requires that the reactions of the enzymes are coordinated so that the rate of synthesis of the intermediate matches its rate of utilization. The linkage between the ATC and DHO subunits was demonstrated by showing that the binding of the bisubstrate analog, N-phosphonacetyl-L-aspartate to the ATC subunit inhibits the activity of the distal DHO subunit. Structural studies identified a DHO loop, loop A, interdigitating between the ATC domains that would be expected to interfere with domain closure essential for ATC catalysis. Mutation of the DHO residues in loop A that penetrate deeply between the two ATC domains inhibits the ATC activity by interfering with the normal reciprocal linkage between the two enzymes. Moreover, a synthetic peptide that mimics that part of the DHO loop that binds between the two ATC domains was found to be an allosteric or noncompletive ATC inhibitor (K(i) = 22 μM). A model is proposed suggesting that loop A is an important component of the functional linkage between the enzymes.

Keywords: CAD; N-phosphonacetyl-L-aspartate; allosteric regulation; aspartate transcarbamoylase; dihydroorotase; intersubunit communication; linkage; metabolic channeling; pyrimidine biosynthesis; thermophile.

Scheme 1

Reactions catalyzed by A. aeolicus DAC.

Figure 1

Structure of the DAC complex. (A) The intact DAC dodecamer showing the arrangement of subunits. The ATC subunits associate to form two trimers, one on the front in this view and the other hidden behind the structure. The DHO subunits are associated as a peripheral ring consisting of three DHO dimers. (B) The large central cavity of the complex (diameter 65 Å) can be seen if the front and back ATC trimers that cap the cavity, are removed. (C) A space-filled view, perpendicular to the three-fold axis, of one ATC monomer with its constituent carbamoyl phosphate binding domain (CP) and aspartate binding domain (ASP) is shown bound to the nearest DHO monomer. The loop on the DHO domain, loop A interdigitates between the two ATC domains. (D) The backbone of the same ATC-DHO pair as in C, showing loop A, the active site histidine, His126, on the ATC subunit and the Zn at the active site of DHO. The distance between the active sites is approximately 14 Å. (E) Three hydrophobic residues of loop A, Ala198^D, Leu199^D and Leu200^D (space filled) are wedged between the two ATC domains; the CP domain, residues 1-150 and the ASP domain, residues 151-304. Most of the loop A neighbors, (space filled) on the ATC domain are hydrophobic. Superscripts A and D indicate residues on the ATC and DHO subunits, respectively. The figure was composed with RasWin using the structural coordinates deposited in the Protein Data Bank (PDB Id: 3B6N).

Figure 2

The effect of PALA bound to ATC on the activity of DHO. The DAC complex was formed by mixing stoichiometric amounts of the purified Aquifex aeolicus DHO and ATC. The dihydroorotate saturation curves of the DHO subunit were determined at different concentrations of the potent ATC inhibitor, PALA; no PALA (▪); 0.1 nM PALA (□); 0.25 nM (•); 0.50 nM (○); 1 nM (▴), 1 μM (▵); 2.5 μM (♦); 5 μM (⋄); 10 μM (▾); 100 μM (▿); 1 mM (◪). The assays were performed as described in Materials and Methods, with 40 μg of DHO and 29.6 μg of ATC for each determination.

Figure 3

Steady state kinetics of DHO loop A mutants. (A) the carbamoyl phosphate saturation curve of the ATC component of DAC reconstituted with wild-type ATC and the wild-type DHO (○) and the DHO mutants, Leu200Arg (•), Leu199Arg (□), and Ala198Arg (▪). (B) The dihydroorotate saturation curve of the DHO component of DAC reconstituted with the wild-type ATC and the mutant DHO subunits (same symbols).

Figure 4

Titration of ATC with wild-type and DHO mutants. (A) The isolated ATC subunit (7 μg) was titrated with wild-type DHO (○) and the Leu200Arg (•), Leu199Arg (□), and Ala198Arg (▪) mutants and the DHO activity was assayed. The DHO subunit is active only when present in a complex with ATC, so the endpoint corresponds to the molar ratio at saturation when all of the ATC is complexed to DHO. (B) Sephacryl S-300 gel filtration of the native DAC complex shows a single high molecular weight peak as determined by the absorbance at 280 nm (not shown) and SDS gel electrophoresis (inset). The peak has a 37 kDa ATC subunit and 49 kDa DHO subunit and both ATC (•) and DHO (○) activities. The Ala198Arg mutant column profile has two distinct peaks as determined by absorbance. The first peak consists of the 37 kDa ATC subunit and has ATC activity (▪) whereas the second peak contains only the inactive DHO subunit.

Scheme 2

Sequence of loop A and the peptide inhibitor.

Figure 5

The effect of Peptide on the activities of DAC. (A) The ATC activity of the isolated ATC subunit (•) and of DAC (○) was assayed as a function of the peptide concentration. (B) The DHO activity of DAC (○) and the isolated DHO subunit (•) was also assayed at the indicated concentration of the peptide.

Figure 6

Steady state kinetics of the ATC subunit at various concentrations of the peptide. (A) Carbamoyl phosphate saturation curves were determined in the absence of the peptide (○) and at 50 μM (•), 100 μM (□), 150 μM (▪) and 250 μM (▵) peptide concentrations. The data were fit to the Michaelis–Menton equation and the steady state kinetic parameters are summarized in Table III. (B) The inhibition constant K_i was determined to be 22 μM from a plot of 1/V_max versus the peptide concentration.

Figure 7

Proposed model for the reciprocal coupling of the ATC and DHO subunits of DAC. Both ATC and DHO can exist in an open conformation (superscript o) that can bind substrates and a closed conformation (superscript c) where catalysis occurs. The coupling is reciprocal in that when the ATC domain is closed, the DHO domain is open and vice versa. (a) The binding of carbamoyl phosphate (CP) and aspartate (ASP) induces domain closure of the ATC subunit (A^c). The DHO remains in the open conformation (D^o). (b) carbamoyl aspartate (Casp) is formed. (c) The ATC subunit opens and carbamoyl aspartate (Casp) is released and diffuses to the DHO active site, which then closes in turn (D^c). The open conformation of ATC binds CP and ASP in preparation of another round of catalysis. (d) The conversion of Casp to dihydroorotate (dho) results in the opening of the DHO domain (D^o) and the release of the final product.