Heterotropic interactions in Escherichia coli aspartate transcarbamylase. Subunit interfaces involved in CTP inhibition and ATP activation.
In Escherichia coli aspartate transcarbamylase, each regulatory chain is involved in two kinds of interfaces with the catalytic chains, one with the neighbour catalytic chain which belongs to the same half of the molecule (R1-C1 type of interaction), the other one with a catalytic chain belonging to the other half of the molecule (R1-C4 type of interaction). In the present work, site-directed mutagenesis was used to investigate the involvement of the C-terminal region of the regulatory chain in the process of feed-back inhibition by CTP. Removal of the two last C-terminal residues of the regulatory chains is sufficient to abolish entirely the sensitivity of the enzyme to CTP. Thus, it appears that the contact between this region and the 240s loop of the catalytic chain (R1-C4 type of interaction) is essential for the transmission of the regulatory signal which results from CTP binding to the regulatory site. None of the modifications made in the R1-C4 interface altered the sensitivity of the enzyme to the activator ATP, suggesting that the effect of this nucleotide rather involves the R1-C1 type of interface. These results are in agreement with the previously proposed interpretation that CTP and ATP do not simply act in inverse ways on the same equilibrium.
Xi
X G
XG
Laboratoire d'Enzymologie, C.N.R.S. Gif-sur-Yvette, France.
van Vliet
F
F
Ladjimi
M M
MM
de Wannemaeker
B
B
de Staercke
C
C
Glansdorff
N
N
Piérard
A
A
Cunin
R
R
Hervé
G
G
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000595:Amino Acid Sequence; D001221:Aspartate Carbamoyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002872:Chromosome Deletion; D003570:Cytidine Triphosphate / Q000494:pharmacology*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D014961:X-Ray Diffraction
D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D003570:Cytidine Triphosphate; D000255:Adenosine Triphosphate; D001221:Aspartate Carbamoyltransferase