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Ligation alters the pathway of urea‐induced denaturation of the catalytic trimer of Escherichia coli aspartate transcarbamylase
Author(s) -
Bromberg S.,
Licata V.J.,
Mallikarachchi D.,
Allewell N.M.
Publication year - 1994
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560030809
Subject(s) - trimer , escherichia coli , denaturation (fissile materials) , urea , chemistry , aspartate carbamoyltransferase , ligation , biochemistry , catalysis , biophysics , biology , microbiology and biotechnology , dimer , organic chemistry , gene , nuclear chemistry
We have examined the pathway and energetics of urea‐induced dissociation and unfolding of the catalytic trimer (C 3 ) 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, c 3 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 ΔG dissociation at 7.5 °C of about 7–10 kcal m −1 per interface. ΔG unfolding 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 − , c 3 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.