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.