Document Type

Article

Publication Date

1-1-1994

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. Copyright © 1994 The Protein Society

Publication Source (Journal or Book title)

Protein Science

First Page

1236

Last Page

1244

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