Co-operative interactions between the catalytic sites in Escherichia coli aspartate transcarbamylase. Role of the C-terminal region of the regulatory chains.
In aspartate transcarbamylase (ATCase) each regulatory chain interacts with two catalytic chains each one belonging to a different trimeric catalytic subunit (R1-C1 and R1-C4 types of interactions as defined in Fig. 1). In order to investigate the interchain contacts that are involved in the co-operative interactions between the catalytic sites, a series of modified forms of the enzyme was prepared by site-directed mutagenesis. The amino acid replacements were devised on the basis of the previously described properties of an altered form of ATCase (pAR5-ATCase) which lacks the homotropic co-operative interactions between the catalytic sites. The results obtained (enzyme kinetics, bisubstrate analog influence and pH studies) show that the R1-C4 interaction is essential for the establishment of the enzyme conformation that has a low affinity for aspartate (T state), and consequently for the existence of co-operativity between the catalytic sites. This interaction involves the 236-250 region of the aspartate binding domain of the catalytic chain (240s loop) and the 143-149 region of the regulatory chain which comprises helix H3'.
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
Piérard
A
A
Glansdorff
N
N
Hervé
G
G
Cunin
R
R
D000495:Allosteric Site; D000595:Amino Acid Sequence; D001221:Aspartate Carbamoyltransferase / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D008961:Models, Structural; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D010957:Plasmids; D011485:Protein Binding; D011487:Protein Conformation; D015183:Restriction Mapping
D046911:Macromolecular Substances; D001224:Aspartic Acid; C013195:sparfosic acid; D001221:Aspartate Carbamoyltransferase; D010746:Phosphonoacetic Acid