Tryptophan residues at subunit interfaces used as fluorescence probes to investigate homotropic and heterotropic regulation of aspartate transcarbamylase

The homotropic and heterotropic interactions in Escherichia coli aspartate transcarbamylase (EC 2.1.3.2) are accompanied by various structure modifica tions. The large quaternary structure change associated with the T to R tra nsition, promoted by substrate binding, is accompanied by different local c onformational changes. These tertiary structure modifications can be monito red by fluorescence spectroscopy, after introduction of a tryptophan fluore scence probe at the site of investigation. To relate unambiguously the fluo rescence signals to structure changes in a particular region, both naturall y occurring Trp residues in positions 209c and 284c of the catalytic chains were previously substituted with Phe residues. The regions of interest wer e the so-called 240's loop at position Tyr240c, which undergoes a large con formational change upon substrate binding, and the interface between the ca talytic and regulatory chains in positions Asn153r and Phe145r supposed to play a role in the different regulatory processes. Each of these tryptophan residues presents a complex fluorescence decay with three to four independ ent lifetimes, suggesting that the holoenzyme exists in slightly different conformational states. The bisubstrate analogue N-phosphonacetyl-L-aspartat e affects mostly the environment of tryptophans at position 240c and 145r, and the fluorescence signals were related to ligand binding and the quatern ary structure transition, respectively. The binding of the nucleotide activ ator ATP slightly affects the distribution of the conformational substates as probed by tryptophan residues at position 240c and 145r, whereas the inh ibitor CTP modifies the position of the C-terminal residues as reflected by the fluorescence properties of Trp153r. These results are discussed in cor relation with earlier mutagenesis studies and mechanisms of the enzyme allo steric regulation.