19F nuclear magnetic resonance studies of fluorotyrosine-labeled aspartate transcarbamoylase. Properties of the enzyme and its catalytic and regulatory subunits.
Aspartate transcarbamoylase labeled with 3-fluorotyrosine was purified from an Escherichia coli strain which was auxotrophic for tyrosine and overproduced aspartate transcarbamoylase upon uracil starvation. The labeled enzyme in which about 85% of the tyrosines were replaced by fluorotyrosine exhibited high enzyme activity that varied in a sigmoidal manner with respect to the aspartate concentration. Also, the labeled enzyme was inhibited by CTP, activated by ATP, and exhibited a 2.6% decrease in sedimentation coefficient upon the addition of the active-site ligand, N-(phosphonacetyl)-L-aspartate. Thus, despite extensive replacement of tyrosines by fluorotyrosine, the modified enzyme was similar to native aspartate transcarbamoylase. The 19F nuclear magnetic resonance spectrum of isolated regulatory subunits labeled with fluorotyrosine consisted of a single peak. Addition of the activator, ATP, or the inhibitor, CTP, caused a loss of intensity at about 61.3 ppm upfield from a trifluoroacetic acid reference and an increase at about 61.5 ppm, but CTP also caused an increase at about 61.0 ppm. Five overlapping resonances were observed in the 19F NMR spectrum of unliganded catalytic subunits containing fluorotyrosine. Although the binding of the bisubstrate analog, N-(phosphonacetyl)-L-aspartate, or the combination of carbamoylphosphate and succinate caused similar disappearances of resonances, the addition of N-(phosphonacetyl)-L-aspartate caused the appearance of resonances not observed with carbamoylphosphate plus succinate. Carbamoylphosphate alone perturbed three or four resonances and the subsequent addition of succinate affected at least two.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001221:Aspartate Carbamoyltransferase / Q000037:antagonists & inhibitors; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D001665:Binding Sites; D003570:Cytidine Triphosphate / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives*
C001245:3-fluorotyrosine; D001224:Aspartic Acid; D014443:Tyrosine; D003570:Cytidine Triphosphate; C013195:sparfosic acid; D000255:Adenosine Triphosphate; D001221:Aspartate Carbamoyltransferase; D010746:Phosphonoacetic Acid