19F nuclear magnetic resonance studies of communication between catalytic and regulatory subunits in aspartate transcarbamoylase.
19F nuclear magnetic resonance (NMR) spectroscopy was used to study "communication" between the catalytic and regulatory subunits in aspartate transcarbamoylase of Escherichia coli. Hybrid enzymes composed of fluorotyrosine-labeled regulatory subunits and native catalytic subunits or of native regulatory subunits and fluorotyrosine-labeled catalytic subunits were constructed and shown to have the allosteric kinetic properties of native enzyme. These hybrids exhibited the ligand-promoted "global" conformational changes characteristic of native aspartate transcarbamoylase and alterations in the NMR spectrum when ligands bind to the active site. The NMR difference spectrum caused by the binding of the bisubstrate analog N-(phosphonacetyl)-L-aspartate to the hybrid containing 19F-labeled regulatory chains consisted of two troughs and a peak, suggesting that two tyrosines in the regulatory polypeptide chains were affected by the binding of ligand to the catalytic subunits. The increase in magnitude of the peak appeared to depend directly on the fractional saturation of the active sites. A peak with two distinct shoulders was observed in the 19F NMR spectrum of the hybrid containing fluorotyrosine in the catalytic chains when it was saturated with the ligand, whereas the spectrum for the unliganded enzyme consisted of a single peak. The NMR difference spectrum showed that the bisubstrate ligand perturbed at least two resonances, and these changes appeared to be tightly linked to the binding of the ligand.
D001221:Aspartate Carbamoyltransferase; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D001665:Binding Sites; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D011487:Protein Conformation; D014443:Tyrosine / Q000031:analogs & derivatives*
C001245:3-fluorotyrosine; D001224:Aspartic Acid; D014443:Tyrosine; C013195:sparfosic acid; D001221:Aspartate Carbamoyltransferase; D010746:Phosphonoacetic Acid