Substitutions in the aspartate transcarbamoylase domain of hamster CAD disrupt oligomeric structure

Abstract

Aspartate transcarbamoylase (ATCase; EC 2.1.3.2) is one of three enzymatic domains of CAD, a protein whose native structure is usually a hexamer of identical subunits. Alanine substitutions for the ATCase residues Asp-90 and Arg-269 were generated in a bicistronic vector that encodes a 6-histidine-tagged hamster CAD. Stably transfected mammalian cells expressing high levels of CAD were easily isolated and CAD purification was simplified over previous procedures. The substitutions reduce the ATCase V(max) of the altered CADs by 11-fold and 46-fold, respectively, as well as affect the enzyme's affinity for aspartate. At 25 mM Mg(2+), these substitutions cause the oligomeric CAD to dissociate into monomers. Under the same dissociating conditions, incubating the altered CAD with the ATCase substrate carbamoyl phosphate or the bisubstrate analogue N-phosphonacetyl-L-aspartate unexpectedly leads to the reformation of hexamers. Incubation with the other ATCase substrate, aspartate, has no effect. These results demonstrate that the ATCase domain is central to hexamer formation in CAD and suggest that the ATCase reaction mechanism is ordered in the same manner as the Escherichia coli ATCase. Finally, the data indicate that the binding of carbamoyl phosphate induces conformational changes that enhance the interaction of CAD subunits.

Figure 1

SDS/PAGE screen for high expressing cell line. The cellular protein from each well of 96-well tissue culture plates was subjected to SDS/PAGE and stained with Coomassie blue. Lane M contains molecular mass markers. Lane 1 is the untransfected G9C cells. Lanes 2–11 are different clones of transfectants. The arrow indicates the band corresponding to CAD, which is observed only in lanes 4–8.

Figure 2

CAD purification. Lanes 1 and 2 are cell-free extracts from an untransfected and CAD-wt-expressing cell lines, respectively. Extract from a CAD-wt-expressing cell line was then purified by Ni²⁺ affinity chromatography. Lane 3 is the flow-through. Lanes 4–6 are the washing steps with increasing concentrations of imidazole: 5 mM, 60 mM, and 100 mM. Lanes 7–10 are eluting fractions with 1 M imidazole. The SDS/PAGE gel was stained by Coomassie blue.

Figure 3

Aspartate kinetic analysis of CAD ATCase. The assays were performed on purified CAD-wt (♦), CAD-D90A (■), and CAD-R269A (▴) in the absence of magnesium. Each point represents the mean ± SD from triplicate determinations. The details of the altered CADs are shown in the Inset.

Figure 4

Sedimentation of glycerol gradient in the absence of effectors. Wild-type CAD (♦), CAD-D90A (■), and CAD-R269A (▴) were sedimented on 8–20% glycerol gradients with 25 mM MgCl₂. The molecular weight standard (●) was run in parallel.

Figure 5

Influence of different effectors to CAD-R269A sedimentation. (A) CAD-R269A sedimented through 4 mM MgCl₂ (⋄) or 25 mM MgCl₂ (♦). (B) CAD-R269A incubated in the presence of 10 μM PALA (■) or in the absence of PALA (♦) before sedimentation and CAD-R269A incubated and sedimented with 1.6 mM aspartate (●) or 2.5 mM carbamoyl phosphate (▴). Mg²⁺ was present at 25 mM in all of the experiments shown in B.