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Protein Science (2002), 11:1074-1081.
Copyright © 2002 The Protein Society

Replacement of Asp-162 by Ala prevents the cooperative transition by the substrates while enhancing the effect of the allosteric activator ATP on E. coli aspartate transcarbamoylase

L. Fetler1,,2,,5, P. Tauc3, D.P. Baker4,,6, C.P. Macol4, E.R. Kantrowitz4 and P. Vachette1

1 Laboratoire pour l'Utilisation du Rayonnement Electromagnétique (CNRS, CEA, MER), Université Paris-Sud, F-91898 Orsay Cedex, France
2 Laboratoire de Biochimie des Signaux Régulateurs Cellulaires et Moléculaires, UMR-CNRS 7631, Université Pierre et Marie Curie, F-75006 Paris, France
3 Laboratoire de Biotechnologies et Pharmacologie Génétique Appliquée, UMR-CNRS 8532, Ecole Normale Supérieure de Cachan, F-94235 Cachan, France
4 Merkert Chemistry Center, Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02467, USA

Reprint requests to: P. Vachette, Laboratoire pour l'Utilisation du Rayonnement Electromagnétique (CNRS, CEA, MER), B|fqtiment 209D, B.P. 34, Université Paris-Sud, F-91898 Orsay Cedex, France; e-mail vachette{at}lure.u-psud.fr; fax +33-1-64-46-41-48.

The available crystal structures of Escherichia coli aspartate transcarbamoylase (ATCase) show that the conserved residue Asp-162 from the catalytic chain interacts with essentially the same residues in both the T- and R-states. To study the role of Asp-162 in the regulatory properties of the enzyme, this residue has been replaced by alanine. The mutant D162A shows a 7700-fold reduction in the maximal observed specific activity, a twofold decrease in the affinity for aspartate, a loss of homotropic cooperativity, and decreased activation by the nucleotide effector adenosine triphosphate (ATP) compared with the wild-type enzyme. Small-angle X-ray scattering (SAXS) measurements reveal that the unliganded mutant enzyme adopts the T-quaternary structure of the wild-type enzyme. Most strikingly, the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA) is unable to induce the T to R quaternary structural transition, causing only a small alteration of the scattering pattern. In contrast, addition of the activator ATP in the presence of PALA causes a significant increase in the scattering amplitude, indicating a large quaternary structural change, although the mutant does not entirely convert to the wild-type R structure. Attempts at modeling this new conformation using rigid body movements of the catalytic trimers and regulatory dimers did not yield a satisfactory solution. This indicates that intra- and/or interchain rearrangements resulting from the mutation bring about domain movements not accounted for in the simple model. Therefore, Asp-162 appears to play a crucial role in the cooperative structural transition and the heterotropic regulatory properties of ATCase.

Keywords: Aspartate transcarbamoylase; small-angle X-ray scattering; allostery; cooperativity; quaternary structural changes

Abbreviations: ATCase, aspartate transcarbamoylase (carbamoyl phosphate: L-aspartate transferase) from E. coli (EC 2.1.3.2.) • [Asp]0.5, the aspartate concentration at half the maximal observed specific activity • PALA, N-(phosphonacetyl)-L-aspartate • 160's loop, loop region in the catalytic chain corresponding to residues 160-166 • 240's loop, loop region encompassing residues 230-245 in the catalytic chain • SAXS, small-angle X-ray scattering • holoenzyme, the entire molecule consisting of six catalytic chains and six regulatory chains • c followed by a number, e.g., c1 or c4, refers to a particular catalytic chain


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