X-ray scattering titration of the quaternary structure transition of aspartate transcarbamylase with a bisubstrate analogue: influence of nucleotide effectors.
The regulation of aspartate transcarbamylase (ATCase) involves various conformational changes, including a large quaternary structure rearrangement. This is directly related to a major change in its solution X-ray scattering curve upon binding the bisubstrate analogue N-(phosphonacetyl)-L-aspartate (PALA), allowing us to monitor directly the amount of the different quaternary structures present in solution. Data were analysed by singular vector decomposition without any prior assumption as to the number of quaternary structure states. Scattering curves in the presence of variable concentrations of PALA, alone or with saturating CTP or ATP, can be accounted for with only two states. Consequently the method gives the fraction of molecules in either state. Whereas CTP slightly decreases the proportion of molecules in the R state, ATP has no detectable effect, whatever the amount of PALA ligated to ATCase. The requirement for only two quaternary structures, suggesting a concerted transition, promoted us to test the ability of the classical model, proposed by Monod, Wyman and Changeux, to account for our data. By and large, it is satisfactory as regards the homotropic effect of PALA and the observed effect of CTP, although it remains incompatible with some other observations, which support the involvement of more indirect mechanisms in the inhibitory properties of CTP. But ATP does not directly influence the T to R transition and consequently must act by a totally different mechanism.
Fetler
L
L
Laboratoire de Biochimie des Signaux Régulateurs, Cellulaires et Moléculaires URA CNRS 1682, Université Pierre et Marie Curie, Paris, France.
Tauc
P
P
Hervé
G
G
Moody
M F
MF
Vachette
P
P
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000494:Allosteric Regulation; D001221:Aspartate Carbamoyltransferase / Q000737:chemistry* / Q000378:metabolism; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D003570:Cytidine Triphosphate / Q000494:pharmacology; D008956:Models, Chemical; D009711:Nucleotides / Q000494:pharmacology*; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D011487:Protein Conformation; D012542:Scattering, Radiation; D014965:X-Rays
D009711:Nucleotides; D001224:Aspartic Acid; D003570:Cytidine Triphosphate; C013195:sparfosic acid; D000255:Adenosine Triphosphate; D001221:Aspartate Carbamoyltransferase; D010746:Phosphonoacetic Acid