title: 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.
abstract: 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 To study the role of Asp-162 in the regulatory properties of the enzyme, this residue has been replaced by alanine.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.
authors.affiliation: Laboratoire pour l'Utilisation du Rayonnement Electromagnétique (CNRS, CEA, MER), Université Paris-Sud, F-91898 Orsay Cedex, France.
authors.forename: L
authors.initials: L
authors.lastname: Fetler
authors.forename: P
authors.initials: P
authors.lastname: Tauc
authors.forename: D P
authors.initials: DP
authors.lastname: Baker
authors.forename: C P
authors.initials: CP
authors.lastname: Macol
authors.forename: E R
authors.initials: ER
authors.lastname: Kantrowitz
authors.forename: P
authors.initials: P
authors.lastname: Vachette
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000494:Allosteric Regulation; D019943:Amino Acid Substitution; D001221:Aspartate Carbamoyltransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001224:Aspartic Acid / Q000031:analogs & derivatives* / Q000378:metabolism*; D004926:Escherichia coli; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D020836:Protein Structure, Quaternary / Q000187:drug effects; D013379:Substrate Specificity; D014965:X-Rays
chemical_list: D001224:Aspartic Acid; C013195:sparfosic acid; D000255:Adenosine Triphosphate; D001221:Aspartate Carbamoyltransferase; D010746:Phosphonoacetic Acid