Protein Science, Vol 3, Issue 8 1236-1244, Copyright © 1994 by Cold Spring Harbor Laboratory Press

ARTICLE

Ligation alters the pathway of urea-induced denaturation of the catalytic trimer of Escherichia coli aspartate transcarbamylase

S. BROMBERG, V. J. LICATA, D. MALLIKARACHCHI and N. M. ALLEWELL
Present address: Department of Pharmaceutical Chemistry, University of California at San Francisco, Laurel Heights Campus, 3333 California Street, San Francisco, California 94118.

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 {Delta}G(dissociation) at 7.5{deg}C of about 7-10 kcal m(-1) per interface. {Delta}G(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.
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