Structural fluctuations in aspartate transcarbamylase: Succinimide quenching and fluorescence depolarization of tryptophan and tyrosine residues

The effects of binding of various effector ligands on the dynamics of aspartate transcarbamylase (ATCase, c₆r₆) and on its regulatory (r₂) and catalytic (c₃) subunits were characterized by examining succinimide quenching of the intrinsic fluorescence, and by measurement of the lifetime-resolved anisotropies. The lifetimes of the tryptophan residues in c₃ and c₆r₆ are about 1.7 ns while those of tyrosine residues in r₂ are 2.7 ns. These lifetimes are not significantly altered by the binding of various substrates, substrate analogs and nucleotides. The effects of ligand binding on the accessibility of both tyrosine and tryptophan residues to the quencher are modest in all cases, though the changes are in the same direction as seen using other physicochemical techniques such as hydrogen exchange (M. Lennick and N.M. Allewell, Proc. Natl. Acad. Sci. U.S.A. 78 (1981) 6759). The tryptophan residues in both c₃ and c₆r₆ are immobilized whereas the tyrosine residues of r₂ have some motional freedom. Ligands have no effect on the immobilized tryplophan residues in c₃ and c₆r₆, while binding of nucleotides to r₂ results in a small decrease in the motional freedom of the tyrosine residues. These results suggest that the protein matrix around the aromatic arnino acids in r₂, c₃ and c₆r₆ is rather rigid and that local effects of ligands on the dynamics of these residues, and that of the surrounding protein matrix, are minor. They are in general agreement with the results of the crystal structure determination (R.B. Honzatko et al., J. Mol. Biol. 160 (1982) 219).