The catalytic site of Escherichia coli aspartate transcarbamylase: interaction between histidine 134 and the carbonyl group of the substrate carbamyl phosphate.
Previous pKa determinations indicated that histidine 134, present in the catalytic site of aspartate transcarbamylase, might be the group involved in the binding of the substrate carbamyl phosphate and, possibly, in the catalytic efficiency of this enzyme. In the present work, this residue was replaced by an asparagine through site-directed mutagenesis. The results obtained show that histidine 134 is indeed the group of the enzyme whose deprotonation increases the affinity of the catalytic site for carbamyl phosphate. In the wild-type enzyme this group can be titrated only by those carbamyl phosphate analogues that bear the carbonyl group. In the modified enzyme the group whose deprotonation increases the catalytic efficiency is still present, indicating that this group is not the imidazole ring of histidine 134 (pKa = 6.3). In addition, the pKa of the still unknown group involved in aspartate binding is shifted by one unit in the mutant as compared to the wild type.
Xi
X G
XG
Laboratoire d'Enzymologie, CNRS, Gif-sur-Yvette, France.
Van Vliet
F
F
Ladjimi
M M
MM
Cunin
R
R
Hervé
G
G
D000595:Amino Acid Sequence; D001221:Aspartate Carbamoyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002221:Carbamyl Phosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology*; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D011485:Protein Binding; D013379:Substrate Specificity; D013386:Succinates / Q000378:metabolism; D019802:Succinic Acid
D001426:Bacterial Proteins; D013386:Succinates; D001224:Aspartic Acid; D006639:Histidine; D002221:Carbamyl Phosphate; C013195:sparfosic acid; D019802:Succinic Acid; D001221:Aspartate Carbamoyltransferase; D010746:Phosphonoacetic Acid