Boundary spreading in sedimentation velocity experiments on partially liganded aspartate transcarbamoylase. A ligand-mediated isomerization.
Transport theory for rapidly reversible interacting systems was used to analyze boundary spreading in sedimentation velocity experiments on partially liganded aspartate transcarbamoylase. In the presence of sub-stoichiometric amounts of a bisubstrate analog, N-(phosphonacetyl)-L-aspartate, which is bound with high affinity to the enzyme (Kd approximately 100 nM), broad boundaries were observed consistent with the presence of two conformational forms. The theoretical treatment showed that under these conditions, the interconversion between the compact (11.7 S) and swollen (11.3 S) forms of the enzyme appears uncoupled, due to the formation of a gradient of free ligand that is caused by the re-equilibration resulting from the differential sedimentation of the two enzyme forms. Sedimentation velocity patterns for such systems are interpretable in terms of two independent species. When, however, the enzyme is in the presence of a sub-saturating amount of the weakly bound ligand, succinate (Kd approximately 1 mM), the re-equilibration caused by the differential sedimentation does not perturb the large background of free ligand and form a gradient. Instead, the two different forms of the enzyme are in dynamic equilibrium, resulting in a boundary having average sedimentation and diffusion coefficients. The observed boundary spreading experiments with different ligands are satisfactorily interpreted in terms of a ligand-mediated isomerization of aspartate transcarbamoylase from a compact to a swollen conformation.
Werner
W E
WE
Department of Molecular Biology, University of California, Berkeley 94720.
Cann
J R
JR
Schachman
H K
HK
D001221:Aspartate Carbamoyltransferase; D001224:Aspartic Acid / Q000031:analogs & derivatives; D001692:Biological Transport; D004926:Escherichia coli; D007536:Isomerism; D008024:Ligands; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives; D014461:Ultracentrifugation
D008024:Ligands; D001224:Aspartic Acid; C013195:sparfosic acid; D001221:Aspartate Carbamoyltransferase; D010746:Phosphonoacetic Acid