A cooperative Escherichia coli aspartate transcarbamoylase without regulatory subunits

Abstract

Here we report the isolation, kinetic characterization, and X-ray structure determination of a cooperative Escherichia coli aspartate transcarbamoylase (ATCase) without regulatory subunits. The native ATCase holoenzyme consists of six catalytic chains organized as two trimers bridged noncovalently by six regulatory chains organized as three dimers, c(6)r(6). Dissociation of the native holoenzyme produces catalytically active trimers, c(3), and nucleotide-binding regulatory dimers, r(2). By introducing specific disulfide bonds linking the catalytic chains from the upper trimer site specifically to their corresponding chains in the lower trimer prior to dissociation, a new catalytic unit, c(6), was isolated consisting of two catalytic trimers linked by disulfide bonds. Not only does the c(6) species display enhanced enzymatic activity compared to the wild-type enzyme, but the disulfide bonds also impart homotropic cooperativity, never observed in the wild-type c(3). The c(6) ATCase was crystallized in the presence of phosphate and its X-ray structure determined to 2.10 A resolution. The structure of c(6) ATCase liganded with phosphate exists in a nearly identical conformation as other R-state structures with similar values calculated for the vertical separation and planar angles. The disulfide bonds linking upper and lower catalytic trimers predispose the active site into a more active conformation by locking the 240s loop into the position characteristic of the high-affinity R state. Furthermore, the elimination of the structural constraints imposed by the regulatory subunits within the holoenzyme provides increased flexibility to the c(6) enzyme, enhancing its activity over the wild-type holoenzyme (c(6)r(6)) and c(3). The covalent linkage between upper and lower catalytic trimers restores homotropic cooperativity so that a binding event at one or so active sites stimulates binding at the other sites. Reduction of the disulfide bonds in the c(6) ATCase results in c(3) catalytic subunits that display kinetic parameters similar to those of wild-type c(3). This is the first report of an active c(6) catalytic unit that displays enhanced activity and homotropic cooperativity.

Figure 1

Characterization of c₆ by gel electrophoresis. Non-denaturating (A) and non-reducing SDS PAGE (B). The two gels were composed of 7.5% and 12% acrylamide, respectively, and only differed in that the non-reducing gel did not have 2-mercaptoethanol added to either gel or loading buffer. Lane 1, wild-type E. coli ATCase holoenzyme; Lane 2, E. coli ATCase catalytic subunit (c₃); Lane 3, C47A/A241C holoenzyme; Lane 4, C47A/A241C after treatment with pHMB; Lane 5–6, Pure C47A/A241C c₆ at a low and high concentration, respectively.

Figure 2

Aspartate saturation curves for the wild-type and mutant enzymes. Specific activity (mmol•hr⁻¹•mg⁻¹) versus the concentration of Asp for the C47A/A241C c₆ (filled circles) and wild-type holoenzyme (c₆r₆) (open circles) enzymes. The assays were performed at 25° C at saturating concentrations of CP (4.8 mM) in 50 mM Tris-acetate buffer, pH 8.3.

Figure 3

Activity of the C47A/A241C c₆ and c₆r₆ enzymes as a function of PALA concentration. Assays were performed at 25° C at saturating concentrations of CP (4.8 mM) in 50 mM tris-acetate buffer, pH 8.3 for the C47A/A241C c₆ (filled circles) and C47A/A241C c₆r₆ (open circles) enzymes. The Asp concentration was held constant at 8 mM for the mutant catalytic subunit and 7 mM Asp was used for the mutant holoenzyme.

Figure 4

Non-reducing 12% SDS PAGE of dissolved crystals of the C47A/A241C c₆ enzyme. A non-reducing SDS PAGE of a single crystal of the C47A/A241C c₆ enzyme washed and dissolved in 100 mM Tris-acetate buffer pH 8.3. Lane 1, wild-type E. coli ATCase holoenzyme; Lane 2, C47A/A241C c₆ solution before crystallization; Lane 3, C47A/A241C c₆ crystal dissolved in buffer.

Figure 5

Disulfide linkages between upper and lower catalytic subunits. Stereoview of the 240’s loop from a single catalytic chain from the upper catalytic trimer, maroon, and a single catalytic chain from the lower catalytic trimer, gold. These two catalytic chains are covalently linked via a disulfide bond formed between the side chains of Cys241 residues in the upper and lower catalytic chains. The refined coordinates of residue Cys241 from both chains are overlaid on the 2F_o–F_c electron density map (blue) contoured at 1.0 σ. The Cys241 residue exists in two alternate conformations, each with 50% occupancy. The sulfur atoms involved in the bonds are green for one conformation and orange for the other. One position of the disulfide bond is outlined with maroon carbons and the other position with gold carbons.

Figure 6

Active site interactions with P_i ligands. Stereoview of the interactions between the active site residues and the phosphate molecules generated by POVScript⁺ The refined coordinates of the backbone and side chains are overlaid on the 2F_o-F_c electron density map (grey) shown contoured at 1.5σ. The two phosphate molecules are overlaid on the composite omit map (magenta) contoured at 1.0σ. Residues labeled with (*) indicate they are donated from an adjacent catalytic chain.

Figure 7

Superposition of the active sites from the c_{6_Pi} and R_{S-S_Pi} structures. Stereoview of the active site superposition of the c_{6_Pi} structure complexed with P_i1 and P_i2 (white carbons) and the R_{S-S_Pi} structure complexed with P_i1 and P_i2 (blue carbons and P_i). This figure was drawn with PyMOL.

Figure 8

Comparison of the c_{6_Pi} and R_PALA structures. Comparison of one catalytic chain from the upper catalytic trimer, C1 maroon, and the corresponding catalytic chain from the lower catalytic trimer, C4 gold, from the c_{6_Pi} and R_PALA structures. The width of the tube is proportional to the RMS deviation between the α-carbon positions of the c_{6_Pi} and R_PALA structures. Regions colored in blue represent a RMS deviation greater than 1.0 Å. The two P_i molecules bound at the active site are shown as spheres distinguishable by the different coloring of the oxygen atoms. This figure was drawn using PyMOL.