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 tryptophan 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 amino 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).
D001221:Aspartate Carbamoyltransferase / Q000378:metabolism*; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology; D008024:Ligands; D046911:Macromolecular Substances; D011485:Protein Binding; D013050:Spectrometry, Fluorescence / Q000379:methods; D013388:Succinimides / Q000494:pharmacology*; D014364:Tryptophan; D014443:Tyrosine
D008024:Ligands; D046911:Macromolecular Substances; D013388:Succinimides; D014443:Tyrosine; D014364:Tryptophan; D001221:Aspartate Carbamoyltransferase