Photoaffinity labeling of rat liver carbamoyl phosphate synthetase I by 8-azido-ATP.
8-Azido-ATP has been found to serve as a photoaffinity label for two distinct ATP sites on rat liver carbamoyl phosphate synthetase I and to allow preliminary localization of these sites. In the dark, 8-azido-ATP acted as a competitive inhibitor with respect to ATP. Ultraviolet irradiation of carbamoyl phosphate synthetase I in the presence of 8-azido-ATP led to an irreversible loss of activity. ATP specifically protected against this inactivation. The incorporation of 2 mol of 8-azido-ATP per mol of enzyme was required for complete inactivation. To localize the 8-azido-ATP-binding sites to discrete regions of carbamoyl phosphate synthetase I which appear to be structural domains, the enzyme was photolabeled with [gamma-32P]8-azido-ATP and subjected to limited proteolytic digestion. The resulting model for the functional roles of the domains is that there is one ATP site on each of the two large internal structural domains of the enzyme. Each of these domains was found to contain the consensus sequences A and B common to many other nucleotide-binding proteins (Walker, J.E., Saraste, M., Runswick, M. J., and Gay, N. J. (1982) EMBO J. 1, 945-951). In addition, there is extensive structural and possibly functional interaction of the smaller N-terminal domain with one of the internal ATP-binding domains, analogous to a subunit interaction observed with the evolutionarily related Escherichia coli carbamoyl phosphate synthetase.
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D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000378:metabolism*; D001665:Binding Sites; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000378:metabolism* / Q000528:radiation effects; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008025:Ligases / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D010777:Photochemistry; D011487:Protein Conformation; D051381:Rats; D014466:Ultraviolet Rays
D000345:Affinity Labels; D001386:Azides; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D008025:Ligases; D002222:Carbamoyl-Phosphate Synthase (Ammonia)