The activation of Escherichia coli aspartate transcarbamylase by ATP. Specific involvement of helix H2' at the hydrophobic interface between the two domains of the regulatory chains.
The regulatory chain of E. coli aspartate transcarbamylase (E.C. 2.1.3.2) is folded into two domains. The allosteric domain harbours the regulatory site where the activator ATP and the inhibitors CTP and UTP bind competitively. The zinc domain ensures the contact with the catalytic chains. The interface between these two domains is hydrophobic, and involves the carboxy-terminal part of the helix H2' of the allosteric domain and several residues of the zinc domain. This structural feature mediates the transmission of the ATP regulatory signal. In the present work, site-directed mutagenesis and molecular modelling were used to investigate the role of specific amino acid residues in this process. The modifications of the hydrophobic core which are expected to alter the position of helix H2' reduce or abolish the sensitivity of the enzyme to ATP. The properties of the mutants and the results of modelling are fully consistent and suggest that a movement of helix H2' is part of the mechanism of activation by ATP. A model is proposed to account for the transmission of the ATP signal from the regulatory site to the interface between the regulatory and catalytic chains.
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Laboratoire de Biochimie des Signaux Régulateurs, Cellulaires et Moléculaires, URA CNRS 1682, Université Pierre et Marie Curie, Paris, France.
De Staercke
C
C
Van Vliet
F
F
Triniolles
F
F
Jacobs
A
A
Stas
P P
PP
Ladjimi
M M
MM
Simon
V
V
Cunin
R
R
Hervé
G
G
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000495:Allosteric Site; D001221:Aspartate Carbamoyltransferase / Q000378:metabolism*; D001665:Binding Sites; D003570:Cytidine Triphosphate / Q000378:metabolism; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D014544:Uridine Triphosphate / Q000378:metabolism
D003570:Cytidine Triphosphate; D000255:Adenosine Triphosphate; D001221:Aspartate Carbamoyltransferase; D014544:Uridine Triphosphate