The pAR5 mutation and the allosteric mechanism of Escherichia coli aspartate carbamoyltransferase.
Mutation pAR5 replaces residues 145'-153' at the C terminus of the regulatory (r) chains of Escherichia coli ATCase by a new sequence of six residues. The mutated enzyme has been shown to lack substrate cooperativity and inhibition by CTP. Solution X-ray scattering curves demonstrate that, in the absence of ligands, its structure is intermediate between the T form and the R form. In the presence of N-phosphonacetyl-L-aspartate, the mutant is similar to the wild type. An examination of the crystal structure of unligated ATCase reveals that the mutated site is at an interface between r and catalytic (c) chains, which exists only in the T allosteric form. A computer simulation by energy minimization suggests that the pAR5 mutation destabilizes this interface and induces minor changes in the tertiary structure of r chains. The resulting lower stability of the T form explains the loss of substrate cooperativity. The lack of allosteric inhibition may be related to a new electrostatic interaction made in mutant r chains between the C-terminal carboxylate and a lysine residue of the allosteric domain.
Cherfils
J
J
Laboratoire de Biologie Physicochimique, Université Paris-Sud, Orsay, France.
Vachette
P
P
Tauc
P
P
Janin
J
J
D000494:Allosteric Regulation; D001221:Aspartate Carbamoyltransferase / Q000235:genetics* / Q000378:metabolism; D003198:Computer Simulation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D014961:X-Ray Diffraction
D046911:Macromolecular Substances; D001221:Aspartate Carbamoyltransferase