Allosteric regulation of aspartate transcarbamoylase. Analysis of the structural and functional behavior in terms of a two-state model

Abstract

The kinetic and physical properties of the allosteric enzyme, aspartate transcarbamoylase from Escherichia coli, have been analyzed according to the two-state model (Monod, J., Wyman, J., and Changeux, J.-P. (1965), J . Mol. Biol. 12, 88). An internally consistent set of calculated parameters accounted quantitatively for the results from diverse experiments including: (a) enzyme kinetics as a function of the concentration of aspartate in the presence of saturating carbamoyl phosphate; (b) gross conformational changes of the enzyme, revealed by the decrease in the sedimentation coefficient and the increase in the reactivity of the sulfhydryl groups of the regulatory subunits, as a function of the extent of saturation of the active sites by the bisubstrate analogue, N-(phosphonacety1)-L-aspartate; (c) stimulation of enzymic activity at low concentrations of the substrate analogue, succinate; and (d) effects of the inhibitor, CTP, and the activator, ATP, on the kinetic and physical properties of the enzyme. The data were interpreted in terms of an equilibrium between a constrained or low-affinity (T) state and a relaxed or highaffinity (R) form of the enzyme and the perturbation of the equilibrium by the addition of various ligands. In the absence

A solution NMR study showing that active site ligands and nucleotides directly perturb the allosteric equilibrium in aspartate transcarbamoylase

“…X-ray results establish that the structure is made up of a dimer of catalytic trimers (c 3 ) along with three copies of regulatory domain dimers (r 2 ) (8). A large body of data on the enzyme has been obtained over the past 40 years that supports the idea that the homotropic effect can be explained by the Monod-WymanChangeux (MWC) model of allostery (9,10). For an oligomeric protein with n binding sites, this model postulates the existence of two conformations in equilibrium, T and R, with the T form having a lower affinity for substrates (11).…”

Ligand-promoted weakening of intersubunit bonding domains in aspartate transcarbamoylase

“…The dissociation constants for the tetramer-dimer equilibria have been evaluated for hemoglobin in both the oxy and deoxy states (4-9), and the changes in the intersubunit contact energies have been related to the functional properties of the protein (10, 11). In this paper we present an approach aimed at providing comparable information for the regulatory enzyme, aspartate transcarbamoylase (ATCase; carbamoyl- (20).…”

“…Very little exchange of either C or R subunits occurred even in the presence of the bisubstrate analog, N-(phosphonacetyl)-L-aspartate (PALA), which promotes the conformational transition of ATCase to the relaxed state (20,21). Studies were conducted, therefore, on the effect of this active-site ligand on the bonding domains in ATCase-like molecules lacking one R subunit (22)(23)(24)(25).…”

“…The dissociation constants for the tetramer-dimer equilibria have been evaluated for hemoglobin in both the oxy and deoxy states (4-9), and the changes in the intersubunit contact energies have been related to the functional properties of the protein (10, 11). In this paper we present an approach aimed at providing comparable information for the regulatory enzyme, aspartate transcarbamoylase (ATCase; carbamoyl- (20).Since the dissociation of ATCase into free subunits could not be measured directly, we initiated studies of subunit exchange as an alternative, sensitive approach for estimating the strengths of the intersubunit bonding domains. Very little exchange of either C or R subunits occurred even in the presence of the bisubstrate analog, N-(phosphonacetyl)-L-aspartate (PALA), which promotes the conformational transition of ATCase to the relaxed state (20,21).…”

“…In this paper we present an approach aimed at providing comparable information for the regulatory enzyme, aspartate transcarbamoylase (ATCase; carbamoyl- (20).…”