Direct photoaffinity labeling of rat liver carbamoyl phosphate synthetase I with ATP.
The adenine subsites of the ATP sites of rat liver carbamoyl phosphate synthetase I have been localized by direct photoaffinity labeling with ATP. The synthetase is known to utilize two molecules of ATP, apparently in mechanistically discrete steps and at separate ATP sites. UV irradiation of the synthetase in the presence of [alpha-32P]ATP resulted in the incorporation of label. Peptide analysis of the ATP-photolabeled synthetase demonstrated that the labeling was extremely selective. To localize the sites of ATP photoincorporation to discrete regions of the synthetase which appear to be structural domains, the enzyme was photolabeled with [alpha-32P]ATP and subjected to limited proteolytic digestion. Consideration of these data indicated that the internal domains B and C were preferentially labeled and that there was lesser, but significant, labeling of the N-terminal domain A. Omission of the required allosteric activator N-acetylglutamate from the photolabeling mixture resulted in an approximately 60% decrease in label incorporation and an accompanying decrease in the extent of label incorporation in domain B. Consideration of these N-acetylglutamate effects, together with previous findings on the effects of the allosteric activator, confirmed the following functional identification of the ATP sites: domain B participates in binding the molecule of ATP involved in bicarbonate activation, whereas domain C participates in binding the molecule of ATP involved in carbamate phosphorylation.
Potter
M D
MD
Department of Biology, Northeastern University, Boston, Massachusetts 02115.
Powers-Lee
S G
SG
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000378:metabolism* / Q000528:radiation effects; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000302:isolation & purification; D010761:Phosphorus Radioisotopes; D011485:Protein Binding; D051381:Rats; D014466:Ultraviolet Rays
D000345:Affinity Labels; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010761:Phosphorus Radioisotopes; D000255:Adenosine Triphosphate; D002222:Carbamoyl-Phosphate Synthase (Ammonia)