Ligation alters the pathway of urea-induced denaturation of the catalytic trimer ofEscherichia coliaspartate transcarbamylase

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Abstract
We have examined the pathway and energetics of urea-induced dissociation and unfolding of the catalytic trimer (C3) of aspartate transcarbamylase from Escherichia coli at low temperature in the absence and presence of carbamyl phosphate (CP; a substrate), N-(phosphonacetyl)-L-Asp (PALA; a bisubstrate analog), and 2 anionic inhibitors, Cl− and ATP, by analytical gel chromatography supplemented by activity assays and ultraviolet difference spectroscopy. In the absence of active-site ligands and in the presence of ATP, c3 dissociates below 2 M urea into swollen c chains that then gradually unfold from 2 to 6 M urea with little apparent cooperativity. Linear extrapolation to 0 M urea of free energies determined in 3 independent types of experiments yields estimates for ΔGdissociation at 7.5 °C of about 7–10 kcal m−1 per interface. ΔGunfolding of dissociated chains when modeled as a 2-state process is estimated to be very small, on the order of ˜2 kcal m−1. The data are also consistent with the possibility that the unfolding of the dissociated monomer is a 1-state swelling process. In the presence of the ligands CP and PALA, and in the presence of Cl−, c3 dissociates at much higher urea concentrations, and trimer dissociation and unfolding occur simultaneously and apparently cooperatively, at urea concentrations that increase with the affinity of the ligand.

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