The tryptophan residues of aspartate transcarbamylase: site-directed mutagenesis and time-resolved fluorescence spectroscopy.
Aspartate transcarbamylase (EC 2.1.3.2) contains two tryptophan residues in position 209 and 284 of the catalytic chains (c) and no such chromophore in the regulatory chains (r). Thus, as a dodecamer [(c3)2(r2)3] the native enzyme molecule contains 12 tryptophan residues. The present study of the regulatory conformational changes in this enzyme is based on the fluorescence properties of these intrinsic probes. Site-directed mutagenesis was used in order to differentiate the respective contributions of the two tryptophans to the fluorescence properties of the enzyme and to identify the mobility of their environment in the course of the different regulatory processes. Each of these tryptophan residues gives two independent fluorescence decays, suggesting that the catalytic subunit exists in two slightly different conformational states. The binding of the substrate analog N-phosphonacetyl-L-aspartate promotes the same fluorescence signal whether or not the catalytic subunits are associated with the regulatory subunits, suggesting that the substrate-induced conformational change of the catalytic subunit is the essential trigger for the quaternary structure transition involved in cooperativity. The binding of the substrate analog affects mostly the environment of tryptophan 284, while the binding of the activator ATP affects mostly the environment of tryptophan 209, confirming that this activator acts through a mechanism different from that involved in homotropic cooperativity.
Fetler
L
L
Laboratoire d'Enzymologie, Centre National de la Recherche Scientifique, Gif-Sur-Yvette, France.
Tauc
P
P
Hervé
G
G
Ladjimi
M M
MM
Brochon
J C
JC
D001221:Aspartate Carbamoyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D002384:Catalysis; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D014364:Tryptophan / Q000032:analysis*
D001224:Aspartic Acid; C013195:sparfosic acid; D014364:Tryptophan; D001221:Aspartate Carbamoyltransferase; D010746:Phosphonoacetic Acid