Resolving the fluorescence response of Escherichia coli carbamoyl phosphate synthetase: mapping intra- and intersubunit conformational changes

Abstract

Carbamoyl phosphate synthetase (CPS) from Escherichia coli is potentially overlaid with a network of allosterism, interconnecting active sites, effector binding sites, and aggregate interfaces to control its mechanisms of catalytic synchronization, regulation, and oligomerization, respectively. To characterize these conformational changes, a tryptophan-free variant of CPS was genetically engineered by substituting six native tryptophans with tyrosines. Each tryptophan was then reinserted, singly, as a specific fluorescence probe of its corresponding microenvironment. The amino acid substitutions themselves result in little apparent disruption of the protein; variants maintain catalytic and allosteric functionality, and the fluorescence properties of each tryptophan, while unique, are additive to wild-type CPS. Whereas the collective, intrinsic fluorescence response of E. coli CPS is largely insensitive to ligand binding, changes of the individual probes in intensity, lifetime, anisotropy, and accessibility to acrylamide quenching highlight the dynamic interplay between several protein domains, as well as between subunits. W213 within the carboxy phosphate domain, for example, exhibits an almost 40% increase in intensity upon saturation with ATP; W437 of the oligomerization domain, in contrast, is essentially silent in its fluorescence to the binding of ligands. Nucleotide and bicarbonate association within the large subunit induces fluorescence changes in both W170 and W175 of the small subunit, indicative of the type of long-range interactions purportedly synchronizing the carboxy phosphate and amidotransferase domains of the enzyme to initiate catalysis. ATP and ADP engender different fluorescence responses in most tryptophans, perhaps reflecting coordinating, conformational changes accompanying the cycling of reactants and products during catalysis.

FIGURE 1

Crystallographic structure of the E. coli CPS heterodimer emphasizing the position of the intrinsic tryptophan residues. Bound ligands include ADP (red), ornithine (yellow), and IMP (orange). Cys269, known to form a thioester adduct with glutamine at the active site of the small subunit, is shown as a magenta, space-filled residue and labeled as Gln. The six, intrinsic tryptophan residues are highlighted as dark blue, “wire” residues. Coordinates taken from 1CE8 of the PDB.

FIGURE 2

Comparison of values for Q_ax (solid bars) and W_ax (open bars) between tryptophan variants of E. coli CPS. Coupling parameters were determined between substrate nucleotide and the regulators ornithine, UMP, or IMP within the (A) full forward ATP_ase reaction, (B) bicarbonate-dependent ATP_ase reaction, and (C) ATP synthesis reaction.

FIGURE 2

Comparison of values for Q_ax (solid bars) and W_ax (open bars) between tryptophan variants of E. coli CPS. Coupling parameters were determined between substrate nucleotide and the regulators ornithine, UMP, or IMP within the (A) full forward ATP_ase reaction, (B) bicarbonate-dependent ATP_ase reaction, and (C) ATP synthesis reaction.

FIGURE 2

Comparison of values for Q_ax (solid bars) and W_ax (open bars) between tryptophan variants of E. coli CPS. Coupling parameters were determined between substrate nucleotide and the regulators ornithine, UMP, or IMP within the (A) full forward ATP_ase reaction, (B) bicarbonate-dependent ATP_ase reaction, and (C) ATP synthesis reaction.

FIGURE 3

Fluorescence emission spectra of wild-type E. coli CPS, each of the single-tryptophan variants, and an equimolar mix of the variants.

FIGURE 4

Percent changes in the integrated area of the fluorescence emission spectra of wild-type E. coli CPS and each of the single-tryptophan variants upon saturation with (A) 10 mM ornithine, (B) 0.1 mM UMP, (C) 0.1 mM IMP, (D) 5 mM ATP, (E) 5 mM ADP, (F) 10 mM glutamine, (G) 30 mM bicarbonate, and (H) 15 mM carbamoyl phosphate. Colored bars correspond to: wild-type CPS , W71 , W213 , W437 , W461 , W170 , and W175 .

FIGURE 5

Relative change in fluorescence intensity upon the addition of the quencher acrylamide for: wild-type CPS (+), W71 (△), W213 (○), W437 (□), W461 (■), W170 (▲), and W175 (•).

FIGURE 6

Absolute changes in the collisional rate constant between acrylamide and tryptophan within wild-type E. coli CPS and each of the single-tryptophan variants upon saturation with (A) 10 mM ornithine, (B) 0.1 mM UMP, (C) 0.1 mM IMP, (D) 5 mM ATP, (E) 5 mM ADP, (F) 10 mM glutamine, (G) 30 mM bicarbonate, and (H) 15 mM carbamoyl phosphate. Colored bars correspond to: wild-type CPS , W71 , W213 , W437 , W461 , W170 , and W175 .

FIGURE 7

Absolute changes in the anisotropy of tryptophan within wild-type E. coli CPS and each of the single-tryptophan variants upon saturation with (A) 10 mM ornithine, (B) 0.1 mM UMP, (C) 0.1 mM IMP, (D) 5 mM ATP, (E) 5 mM ADP, (F) 10 mM glutamine, (G) 30 mM bicarbonate, and (H) 15 mM carbamoyl phosphate. Colored bars correspond to: wild-type CPS , W71 , W213 , W437 , W461 , W170 , and W175 .