Ligation alters the pathway of urea-induced denaturation of the catalytic trimer of Escherichia coli aspartate transcarbamylase.
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 delta Gdissociation at 7.5 degrees C of about 7-10 kcal m-1 per interface. delta Gunfolding 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.
Bromberg
S
S
Department of Biochemistry, University of Minnesota, St. Paul 55108.
LiCata
V J
VJ
Mallikarachchi
D
D
Allewell
N M
NM
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000838:Anions; D001221:Aspartate Carbamoyltransferase / Q000737:chemistry* / Q000378:metabolism; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D001665:Binding Sites; D002221:Carbamyl Phosphate / Q000494:pharmacology; D002384:Catalysis; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002712:Chlorides / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D046911:Macromolecular Substances; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D011489:Protein Denaturation; D017510:Protein Folding; D013053:Spectrophotometry; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology*
D000838:Anions; D002712:Chlorides; D046911:Macromolecular Substances; D001224:Aspartic Acid; D002221:Carbamyl Phosphate; C013195:sparfosic acid; D000255:Adenosine Triphosphate; D014508:Urea; D001221:Aspartate Carbamoyltransferase; D010746:Phosphonoacetic Acid