Direct observation in solution of a preexisting structural equilibrium for a mutant of the allosteric aspartate transcarbamoylase

doi:10.1073/pnas.0607641104

Direct observation in solution of a preexisting structural equilibrium for a mutant of the allosteric aspartate transcarbamoylase

*Centre de Recherche, Institut Curie, F-75248 Paris, France;
^†Laboratoire Physico-Chimie, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 168, F-75248 Paris, France;
^‡Department of Chemistry, Boston College, Merkert Chemistry Center, Chestnut Hill, MA 02467; and
^§Institut de Biochimie et Biophysique Moléculaire et Cellulaire, Unité Mixte de Recherche 8619, Centre National de la Recherche Scientifique, Université Paris-Sud, Bâtiment 430, F-91405 Orsay Cedex, France

Edited by Axel T. Brunger, Stanford University, Stanford, CA, and approved November 13, 2006 (received for review September 1, 2006)

Abstract

Many signaling and metabolic pathways rely on the ability of some of the proteins involved to undergo a substrate-induced transition between at least two structural states. Among the various models put forward to account for binding and activity curves of those allosteric proteins, the Monod, Wyman, and Changeux model for allostery theory has certainly been the most influential, although a central postulate, the preexisting equilibrium between the low-activity, low-affinity quaternary structure and the high-activity, high-affinity quaternary structure states in the absence of substrates, has long awaited direct experimental substantiation. Upon substrate binding, allosteric Escherichia coli aspartate transcarbamoylase adopts alternate quaternary structures, stabilized by a set of interdomain and intersubunit interactions, which are readily differentiated by their solution x-ray scattering curves. Disruption of a salt link, which is observed only in the low-activity, low-affinity quaternary structure, between Lys-143 of the regulatory chain and Asp-236 of the catalytic chain yields a mutant enzyme that is in a reversible equilibrium between at least two states in the absence of ligand, a major tenet of the Monod, Wyman, and Changeux model. By using this mutant as a magnifying glass of the structural effect of ligand binding, a comparative analysis of the binding of carbamoyl phosphate (CP) and analogs points out the crucial role of the amine group of CP in facilitating the transition toward the high-activity, high-affinity quaternary state. Thus, the cooperative binding of aspartate in aspartate transcarbamoylase appears to result from the combination of the preexisting quaternary structure equilibrium with local changes induced by CP binding.

Footnotes

^¶To whom correspondence should be addressed. E-mail: patrice.vachette{at}ibbmc.u-psud.fr
Author contributions: L.F. and P.V. designed research; L.F. and P.V. performed research; E.R.K. contributed new reagents/analytic tools; L.F. and P.V. analyzed data; and L.F. and P.V. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS direct submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0607641104/DC1.
Abbreviations:

ATCase,

aspartate transcarbamoylase (aspartate carbamoyltransferase) from E. coli (EC 2.1.3.2.);

CP,

carbamoyl phosphate;

PALA,

N-(phosphonacetyl)-l-aspartate;

T,

low-activity, low-affinity quaternary structure state;

R,

high-activity, high-affinity quaternary structure state;

MWC model,

Monod, Wyman, and Changeux model for allostery;

SAXS,

small-angle X-ray scattering.